title: Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real-time PCR, immunofluorescence and ELISA methods.
abstract: Chlamydia pneumoniae is an intracellular gram-negative bacterium, which replicates only in eukaryotic cells. Quantification of C. pneumoniae in cell culture is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally, this has been performed by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary, and multiple inclusions are also seen. Therefore, methods are needed to quantify exact amounts of C. pneumoniae in cells. Here, we describe a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes in cultures. In human epithelial (HL) cell cultures, the C. pneumoniae inclusion numbers and the ratio of C. pneumonia genomes/human genome (Cpn/Hum) correlated significantly (r = 0.978, p < 0.001); thus with HL cells, both methods are usable. However, in macrophage cultures, the correlation was weaker (r = 0.133, p = 0.036) and we recommend PCR quantification for exact measurements.
authors.affiliation: Department of Medical Microbiology, Institute of Diagnostics, University of Oulu, Oulu, Finland. kari.poikonen@oulu.fi
authors.forename: Kari
authors.initials: K
authors.lastname: Poikonen
authors.forename: Taina
authors.initials: T
authors.lastname: Lajunen
authors.forename: Sylvi
authors.initials: S
authors.lastname: Silvennoinen-Kassinen
authors.forename: Maija
authors.initials: M
authors.lastname: Leinonen
authors.forename: Pekka
authors.initials: P
authors.lastname: Saikku
mesh_terms: D023521:Chlamydophila Infections / Q000175:diagnosis / Q000382:microbiology*; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D016680:Genome, Bacterial; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis; D008264:Macrophages / Q000382:microbiology*; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D018709:Statistics, Nonparametric
chemical_list: D004269:DNA, Bacterial; D008070:Lipopolysaccharides