For many years researchers have exploited the ability to see Ct inclusions under light microscope for infectivity assay. The number of inclusions formed in host cells can be calculated with the naked eye under a microscope (Figure 1). Chlamydia EB stocks can be used to infect epithelial cells lines . As there is one-to-one relationship between EB and inclusion at low MOIs, the numbers of inclusions formed in the host cells are counted under light microscope in a given area . If the number of host cells is known, then the percentage of infection and infectivity titer of Chlamydia can be calculated using serial dilutions. The precision and accuracy of this method, however, depends on the number of inclusions per field and the number of fields counted. [₄]

Figure 1

Figure 1: Digital photographs taken with light microscopy. Hela Cells infected with Ct at 24 hours post infection

The main limitation of this method is that it can only be used for Chlamydia serovars which have visible inclusions under light microscope e.g Ct serovar E, so this method cannot be used for serovars which make smaller inclusions e.g. Serovar D. Moreover, inclusion counting cannot be carried out at earlier time points (<20 hours post infection) as the inclusions are too small to be seen under light microscope. Also sometime inclusions can be confused with host cell vacuoles leading to over estimation of infectivity titer. Misidentification of inclusions can result in significant discrepancies in the final result.

Chlamydia specific FITC labeled Anti Lps can also be carried out to determine the number of cells infected with Ct. This method that has been used to quantify chlamydial infection since the early 70s. The inclusions can be visualized under fluorescent microscope and can be quantified and infectivity can be calculated by percentage of host cells infected in a given number of cells. [₅] Figure 2

Figure 2

Figure 2 : DIF antibody labeling. Red indicates Ct , whilein green are Hela Cells

This method eliminates the major drawbacks of light microscopy; Ct with smaller inclusions can be assayed and vacuoles can be distinguished from inclusions. Non-specific florescence, background florescence and longer experiment time are major drawbacks of this method. In addition the precision and accuracy of this method, however, depends on the number of inclusions per field and the number of fields counted- similar to under light microscopy.

To over come the problem of limitation of the visual counting under light microscope and Fluorescent microscope Hori et al. in 2000 devised a simple and rapid method for counting a large number of inclusions using an image analysis system and proposed a sampling method based on a statistical analysis of the data obtained with 84 microscopic fields [₄]. This technique also defines sampling methods for different MOIs.

In this method EB suspensions is mixed with equal volumes of latex nanospheres of known concentration. Samples were fixed to copper grids, stained with uranyl acetate, and examined under EM. Latex spheres and EBs were counted in several fields and the EB concentrations were calculated from the ratio of EBs to latex spheres. Percentage of infection can also be determined [₆]. EM is very accurate method, but has potential limited availability. The processing of sample for EM is time consuming and expensive.

Another tried and tested method is using cytometry for Ct infectivity assay and titration[₇]. HeLa cells plated in monolayers are infected with serial dilutions of EBs and at 24 hours post infection, cells can be trypsinized and fixed, permeabolized and stained using FITC conjugated Ct specific anti LPS antibody Flow cytometry analysis gives a objective quantification of the infection based on fluorescent emitted by FITC conjugated antibody.

The main advantage of flow cytometric counting is that it is quantitative test and there is no user bias. Like any other technique, user-training can is important. A larger number of cells are needed for FACS analysis in addition to expensive equipment , technical for and high running costs.

Ligase chain reactions (LCR) have been recently reported for the quantification of Ct infection[₈]. LCR conditions routinely used for C. trachomatis detection are designed to maximize sensitivity and do not provide quantitative information about the concentration of C. trachomatis. This method can be used for direct quatification from clinical specimen too. Variation in copy number for the plasmid containing the LCR target is also a potential source of error in this assay. Similarly Real Time-PCR can also be used for quantification and discrimination of Ct serovars[₉]. Nucleic acid based quantification has primary application for clinical purpose- as the infected specimen can be directly used for LCR and for discriminating between serovars for epidemiological purposed.