Laboratory methods

- Cell culture:

- Nucleic acid detection in clinical specimens:
* Amplified nucleic acid assay:

Culture detects only viable infectious chlamydial elementary bodies and has minimal potential for contamination. With the advent of DNA amplification techniques, culture tests are used less frequently and are generally now performed only in specialized reference laboratories.
Culture is performed by inoculating specimens onto cell culture monolayers. If sufficient numbers of viable chlamydial elementary bodies are present, they infect the cells and grow to form intracytoplasmic inclusions.
Cell monolayers for culture of C.trachomatis are grown in dram or shell vials on glass coverslips or in the wells of multiwell cell culture dishes. Traditionally, Mc Coy and HeLa 229 cells have been use to support the growth of C.trachomatis. The susceptibility of HeLa 229 cells to infection is increased by pretreatment with DEAE-dextran. The same effect is seen with uncentrifuged Mc Coy cell monolayers.
Before the inoculation is made, clinical specimens should be sonicated to disrupt host cells and inclusions and to separate elementary bodies. If a sonicator is not available, specimens should be vortex thoroughly or can be disrupted with glass beads.
To inoculate the cell cultures, the overlying culture medium should be first be removed and replace with enough of the specimen in culture transport medium to cover the monolayer and prevent drying during subsequent centrifugation. Shell vials are centrifugated to enhance culture sensitivity for 1hour at 1,000 to 3,000 x g temperatures that can range from room temperature to 37°C. The potential for a cytotoxic effect of the specimen on the monolayer is minimized by removal of the residual specimen after centrifugation by aspiration and overlaying the monolayer with fresh cell culture medium. Once inoculated, host monolayers should be incubated in growth medium containing cycloheximide (0.5 to 1.5 mg/ml) to selectively inhibit host cell protein synthesis. Inoculated monolayers are then incubated for 48 to 72 hours at 37°C. After incubation, the medium is removed and the monolayers are fixed with methanol.
The inclusions are visualized following incubation by staining with fluorescently labeled antibodies that bind chlamydial lipopolysaccharide (LPS) to recognize all chlamydial species, or major outer membrane protein (MOMP) for C.trachomatis-specific recognition. The preferred method for identification of inclusion is to stain infected monolayers with species-specific, anti-MOMP fluorescein-labeled monoclonal antibodies.
Some stains as Gram, iodine or Giemsa have been used to visualize chlamydial inclusion in cell culture, but these are not commonly used today due to lack of sensitivity and specificity compared to fluorescent-antibody staining.

The antigen detection assays (nonculture) commercially available for the detection of C.trachomatis in clinical specimens utilize either monoclonal or polyclonal antibodies directed against Chlamydia genus-specific LPS or specific or Chlamydia specific MOMPs as capture or detector reagents. One of several types of chemical labels is conjugated to the detector antibody or antibodies in the assay. Fluorescein isothiocyanate is conjugated to the detector in the DFA method, whereas various enzymes may be used in the EIA or microparticle immunoassays (MEIA) in which 96-well plates, beads, or microparticles are used to trap the C.trachomatis antigens. Depending on the substrate used for the enzyme to act on, the signal measured in the EIAs is a color change or chemiluminescence. All of these nonculture antigen methods have similar sensitivities and specificities when compared to the cell culture.

Serological assays are valuable epidemiology tools and can help in the diagnosis of systemic infections such as pneumonia, ectopic pregnancy or tubal factor infertility where antibody titers are often elevated.
There are serological assays that detect antibodies common to all members of the genus. The most common are complement fixation (CF), recombinant enzyme-linked immunofluorescence (rELISA), and whole inclusion immunofluorescence (WIF), which are mainly based on the detection of LPS.

Detects complement-fixing antibodies that recognize the genus-specific LPS antigen and is not specific for any one chlamydial species. Treatment with antibiotics can delay or dismiss the production of CF antibody and will reduce the sensitivity of the test.

This test detect reactivity to genus-specific antigen, or LPS, of chlamydial elementary and reticulate bodies.Now, these tests are commercially available.

The MIF test is the most sensitive of the serologic tests for Chlamydia species and the only serologic test that detects species-specific responses. It is based on the visualization of elementary bodies or reticulate bodies instead of intact inclusions. This assay, which can measure responses to subclasses IgM, IgA, and IgG, is technically demanding, requiring a well-trained and experienced reader, and thus should be performed only in highly specialized laboratories.

The development of tests based on nucleic acid amplification technology has been the most important advance in the field of chlamydial diagnosis since in vitro cell culture techniques replaced the yolk sac for culture and isolation of the organism from clinical specimens.
Nucleic acid amplification is exquisitely sensitive, capable of detecting as little as a single gene copy, and highly specific. It offers the opportunity to use noninvasive sampling techniques to screen for infections in asyntomatic individual who would not ordinarily seek clinical care.

- PCR (Polymerase chain reaction): The PCR test employs two synthetic oligonucleotide primers (15-30 base pairs) with sequences that are complementary to flanking regions of a specific DNA segment present in the target organism. PCR can be genus, species, group, or strain specific depending on the primer design. A target DNA template in clinical specimens must be made available to the primers by detergent-or heat-mediated lysis of organisms and denaturation of double-stranded DNA. Once the primers are hybridized to the DNA template (one per DNA strand), they are extended into DNA products of a length determined by the distance between the two primer annealing sites. The primer are extended through the activity of a thermostable DNA polymerase enzyme, most commonly Taq polimerase (come from Thermus aquaticus bacteria). The double-stranded PCR products become templates for a second round of primer annealing upon denaturation. Multiple cycles of denaturation, annealing, and extention of products result in a logarithmic amplification of the DNA target segment. The PCR products, also called amplicons, are detected by electrophoresis and staining with a DNA intercalating fluorescent dye as ethidium bromide.

- LCR (Ligase chain reaction): The LCR test employs four synthetic oligonucleotide probes (two per DNA strand) anneal at specific target sites on the cryptic plasmid. Each pair of probes hybridize close together on the target DNA template, with 1- to 2- nucleotide gap between. Once the probes are annealed, the gap is filled by DNA polimerase and close by the ligase enzyme.. This two-step process of closing the gap between annealed probes makes the LCR, in theory, more specific than PCR technology. The ligated probe pairs anneal to each other and, upon denaturation, from the template for successive reaction cycles, thus producing a logarithmic amplification of the target sequence. Like PCR, LCR is made in a thermocycler. The LCR product is detected in an automated instrument that uses an immunocolorimetric bead capture system. At the end of the LCR assay, amplified products are inactivated by the automatic addition of a chelated metal complex and a oxidizing agent.

Currently, there is only a single commercially available DNA probe for detection of C. trachomatis (Gen-Probe). This test employs a chemiluminiscent DNA probe. The probe hybridizes to a species-specific sequence of chlamydial 16s rRNA. Once the DNA-rRNa hybrid is formed, it is adsorbed onto a magnetic bead and the chemiluminiscent response is detected quantitatively with a luminometer. Since actively dividing Chlamydiae containing up to 10e4 copies of 16s rRNA, this test should theoretically be more sensitive than antigen detection systems.