A Comparison of Fluorescent Microscopy Methods for the Detection of Chlamydia trachomatis

Chlamydia trachomatis (C. trachomatis) is the most common bacterial sexually transmitted pathogen worldwide, especially in low- and middle-income countries, including South Africa. Although frequently asymptomatic, C. trachomatis infections in women cause genital inflammation. Given that genital inflammation increases women’s risk for human immunodeficiency virus (HIV) infection, treating and preventing chlamydia is vital. Thus, the development of a vaccine is paramount in controlling infection and spread. Research into new drugs should also be pursued, especially with the rise of antimicrobial resistance. Although C. trachomatis microbiology has advanced with technology, the culture and detection of C. trachomatis in vitro remains challenging. The aim of this study was to establish a method to quantify laboratory adapted stocks of C. trachomatis in in vitro cell culture.

First, C. trachomatis serovars E, H, L1 and L2 were propagated in McCoy cells, using centrifugation using an established protocol. These stocks of unknown concentration were used to compare and optimise quantification methods using fluorescent microscopy. Three commercially available reagents were compared: (1) Pathfinder’s C. trachomatis monoclonal antibody, (2) Invitrogen’s C. trachomatis MOMP antibodies, and (3) Trinity Biotech MicroTrak C. trachomatis culture confirmation kit.

The Pathfinder C. trachomatis monoclonal antibody kit and Invitrogen’s C. trachomatis MOMP Monoclonal Antibody kits both had poor sensitivity and gave high background fluorescent signals. Invitrogen’s polyclonal antibody yielded inconsistent results, being either very weakly fluorescent or giving extremely bright signals. Thus, enumeration using this polyclonal antibody had limited success and results were not reproducible. MicroTrak’s kit, in contrast, allowed for clear visualisation of inclusions and allowed for consistent and successful enumeration, although the manufacturer has since taken the kit off the market.

In the research setting, fluorescent microscopy techniques are widely used for quantification of C. trachomatis due to their high sensitivity and inclusion bodies are easy to distinguish, even with an untrained eye. However, this study showed inconsistent and/or unreliable results from the available kits evaluated. Although, rarely used in a research setting, future studies could explore qPCR-based methods to quantify C. trachomatis in vitro.