Quantification of Chlamydia trachomatis Elementary Bodies in Urine by Ligase Chain Reaction

Abstract

Modification of the standard Chlamydia trachomatis ligase chain reaction (LCR) detection assay resulted in a quantitative test. Sample rates from C. trachomatis standards ranging from 32 to 1,048,576 elementary bodies (EB)/ml of urine exhibited a linear correlation with concentration. Quantitative LCR (qLCR) was used to measure the number of EB per milliliter in 158 urine samples from women in Madagascar that tested positive for C. trachomatis by the standard LCR detection assay. C. trachomatis concentrations displayed an apparent bimodal distribution, with approximately one-third of samples (37%) in a peak ranging from 32 to 1,015 EB/ml (median ‫؍‬ 297 EB/ml) and the remainder (63%) in a grouping with relatively higher concentrations, ranging from 1,086 to 218,670 EB/ml (median ‫؍‬ 7,389 EB/ml). qLCR will be useful for studies of chlamydial infection aimed at understanding the associations of organism burden with clinical manifestations and transmission.Sexually transmitted infection with Chlamydia trachomatis represents a tremendous burden of disease in the United States. Because the organism is difficult to culture, there is little information on the relationship of organism burden to the epidemiology of chlamydial infection and its effect on human health. Tissue culture methods, antigen detection tests, and DNA probe testing have poor sensitivity for chlamydial detection in clinical specimens. Recent advances in nucleic acid amplification techniques have provided more-sensitive tests to detect the presence of C. trachomatis infection. In addition, they allow the use of urine rather than genital secretions for detection. However, these tests are not designed to accurately quantify chlamydia in clinical specimens.Previous attempts to quantify C. trachomatis infection employed quantitative culture. Most assays rely on an indirect estimation of an infected individual's organism burden by a count of the number of chlamydial inclusion-forming units in a monolayer of tissue culture cells. These studies have been limited by the relative insensitivity of culture (9), variability of infectivity of host cells under different culture conditions (7, 11), and an inability to adequately quantify a broad range of organism concentration.Despite these limitations, several studies suggest that some broad epidemiological factors are related to the quantity of C. trachomatis present (1, 2, 4, 6, 10). While younger age, Caucasian race, oral contraception use, low secretory immunoglobulin secretion and cervical ectopy appear to be related to higher organism burdens, the associations of Chlamydia quantities with gender and concurrent infection with Neisseria gonorrhoeae are variable. A recent study demonstrated a relationship between chlamydial inclusion-forming units and clinical manifestations of disease, such as the degree of cervicitis or pelvic inflammatory disease in women and urethritis in men (5). More sensitive detection and quantification methods are needed to guide public health interventions and answer key epidemiologic...

Surface enhanced Raman spectroscopy of Chlamydia trachomatis and Neisseria gonorrhoeae for diagnostics, and extra-cellular metabolomics and biochemical monitoring

“…Interestingly, on Au substrates this cell surface vibrational signature is dominated by protein aggregates but on Ag, this aggregate spectrum is absent and only specific residue and backbone features are detected. Chlamydial loads determined by genotype-based techniques in first-void urine specimens in infected patient range from ~10 1 to 10 5 EB/mL, and ~10 4 EB/mL in vulvo-vaginal swabs 58 , 59 . Therefore the demonstrated diagnostic sensitivity of SERS for chlamydial EB identification (10 2 –104 ifu/mL) falls within this range of EB concentrations in patient samples.…”

One-step purification and concentration of DNA in porous membranes for point-of-care applications

“…Additionally, different infections present at a highly variable pathogen loads. For example, clinical studies have quantified active chlamydia infections in urine at 10 1 -10 5 elementary bodies per mL, 75 Ebola in serum at 10 3 -10 9 RNA copies per mL, 76 and influenza in nasopharyngeal wash at 10 3 -10 7 TCID50 per mL. 77 Each of these infections would benefit from a combination of both target purification and concentration.…”

A modified method for rapid quantification of Chlamydia muridarum using Fluorospot

“…Various methods, i.e. , 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.…”