Laboratory Diagnosis - Chlamydia trachomatis Infections

Laboratory Diagnosis

Various approaches are there for laboratory diagnosis of chlamydial infections as mentioned below. The sensitivity of these methods, however, depends on (a) the nature of the disease, (b) site of infection from where the specimen is collected, and (c) the population of the patient examined.

◗ Specimens

Specimens from urethra, cervix, rectum, oropharynx, and conjunctiva are the frequently collected specimens. In addition, other specimens such as, blood, respiratory secretions, sputum, lung, and other tissues are collected and examined. Pus from bubo is also useful for diagnosis of LGV.

◗ Microscopy

Laboratory diagnosis of chlamydial infection is made by micro-scopic examination of various clinical specimens for dem-onstration of chlamydial inclusion bodies. These inclusion bodies can be demonstrated in specimens stained by Giemsa, Castaneda, Machiavello, or Gimenez stains. C. trachomatis infec-tions of conjunctiva, urethra, and cervix are diagnosed by demonstration of typical reniform inclusion bodies surrounding the nucleus in the stained smear of conjunctiva, urethra, and cervical smears.

◗ Culture

Isolation of C. trachomatis in cell cultures is the more spe-cific method for diagnosis of C. trachomatis infection. Clinical specimens are inoculated in different cell lines for isolation ofChlamydia. The sensitivity of the cell cultures for isolation of C. trachomatis is increased by (a) pretreatment with cycloheximide(i.e., a metabolic inhibitor, which inhibits the metabolism of host cells) and (b) use of irradiated cell lines (treated McCoy cells are most commonly used cell lines for isolation of C. trachomatis).

C. trachomatis infection in cell culture is demonstrated by thepresence of intracellular inclusion bodies. These are detected by the use of iodine stains or fluorescence-conjugated antibod-ies. The culture methods are difficult and expensive. These are specifically preferred for isolation of C. trachomatis from rectal specimens, because noncultural methods are usually negative. The culture shows a sensitivity of 50–90% and specificity of 99%.

◗ Antigen detection

Chlamydial antigen can be detected in clinical specimens by direct fluorescent antibody (DFA) staining and enzyme immu-noassay (EIA). Antibodies prepared against either theChlamydia MOMP or the cell wall LPS are used to detect antigens in clinical specimens by these two methods.

Both DFA staining and EIA are approximately 80% sensitive and 95% specific. Both the methods, however, are labor inten-sive and require trained personnel.

◗ Serodiagnosis

Serodiagnosis is based on detection of antibodies against C. trachomatis in serum. Patients with LGV show a very highlevel of serum antibodies. Antibody-based tests are useful for diagnosis of LGV. CFTs, microimmunofluorescence (MIF), and ELISA are employed to detect specific antibodies in the sera:

·           CFT uses a genus-specific LPS antigen for detection of anti-bodies. A single serum showing a high antibody titer of 1:256 or more, or a fourfold increase in antibody titer of a paired sera sample is highly suggestive of LGV.

·           MIF test is a specific test, which utilizes the species- andserovar-specific antigens, such as Chlamydia MOMPs. Con-firmation of LGV is carried out by the MIF test.

·           ELISA is a genus-specific test that uses LPS antigen like CFT.

The antibodies-based serological tests are of limited value in diagnosis of urogenital infections caused by C. trachomatis. This is because antibody titers cannot differentiate between recent and past infection, since antibodies are present in circulation for a long period of time. Detection of IgM antibody by ELISA, however, is very useful for diagnosis of Chlamydiapneumonia in infants.

◗ Frei’s skin test

Frei’s test is an intradermal skin test used for the diagnosis of LGV. A heat-inactivated C. trachomatis LGV serovar grown in the yolk sac of embryonated egg is used as antigen and 0.1 mL of anti-gen is injected intradermally in the forearm and a control antigen prepared from uninfected yolk sac is injected in the other forearm.

        Frei’s skin test is a delayed hypersensitivity reaction in which positive reaction is shown by development of an inflammatory macule, measuring . 7 mm in diameter, on the test arm after 2 days. The nodule reaches maximum size within 4–5 days. The skin test becomes positive 2–6 weeks after infection and remains positive for several years. However, nowadays skin test is rarely used for diagnosis.