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.

A modified method for rapid quantification of Chlamydia muridarum using Fluorospot

“…, PCR, ELISA, and flow cytometry have been employed for quantification of chlamydial IFU [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]]. While these techniques are available and in use, the inability of PCR to discriminate between active or residual infection [20]; the high detection threshold limit required for ELISA [16]; the inability of flow cytometry to provide accurate information regarding size and structure of chlamydial IFU or its location within a cell [17], and the labor intensive nature of immunochemical approaches [16] requiring highly trained technical personnel underscores the need for alternative approaches. We report here a modified, alternative, Fluorospot assay for visual inspection of infected host cells with the added capability of concurrent traditional enumeration allowing comparison of newly derived automated Fluorospot findings to previously well-established manually based Chlamydia norms.…”

Naked-Eye Enumeration of Single Chlamydia pneumoniae Based on Light Scattering of Gold Nanoparticle Probe

“…Additionally, serological tests for C. pneumoniae, complement fixation (CF), whole-inclusion fluorescence, and enzyme-linked immunosorbent assay (ELISA) all require a very clean environment to avoid false-positive results . Therefore, there is an urgent demand to develop an ultrasensitive, quick, and simple strategy to meet the needs of on-site clinical detection.…”

“…15 Additionally, serological tests for C. pneumoniae, complement fixation (CF), whole-inclusion fluorescence, and enzymelinked immunosorbent assay (ELISA) all require a very clean environment to avoid false-positive results. 16 Therefore, there is an urgent demand to develop an ultrasensitive, quick, and simple strategy to meet the needs of on-site clinical detection. With the development of nanotechnology in recent years, functionalized nanomaterials including magnetic nanomaterial, 17,18 carbon nanomaterial, 19,20 silver nanomaterial, 21,22 and gold nanomaterial 23 were employed to develop methods for pathogen assays.…”

Development of a novel chlamydia growth-monitoring method and its application for screening anti- and pro-chlamydial compounds

“…Considering the previously described methods [95], the rapid estimation of chlamydial growth can be achieved via two approaches. The first approach uses either a fluorimeter or a spectrophotometer to measure the total intensity of a Chlamydia-specific fluorescently labeled antibody [96] or indirectly measure Chlamydia growth by measuring decreased host cell metabolism after Chlamydia-induced lysis [93].…”