Lab Diagnostic Testing: Chlamydia

Conjunctivitis due to chlamydia can be an acute or chronic disease. Chlamydia trachomatis is an intracellular parasite that can be diagnosed in the laboratory with 1) smears (giemsa, immunofluoresence), 2) cell culture, 3) serology, and 4) polymerase chain reaction (PCR). Although our laboratory has utilized all these tests at some time, we currently test for chlamydia with the giemsa stain and PCR. Other laboratories may favor other tests, but we suggest that ophthalmologists only choose tests that are 100 percent specific (avoid positive tests that are actually negative), and highly sensitive (positive results are truly positive). Testing with cell culture should be avoided if PCR is available.

Smears (Giemsa)
PCR

Smears (Giemsa)
Smear specimens should be obtained with a conjunctival spatula. NEVER USE A SWAB (soft-tipped applicator) TO OBTAIN A SMEAR FOR CYTOLOGY! After applying topical anesthetic, specimens are directly collected by firmly scraping the exposed conjunctiva. These specimens are transferred to glass microscope slides, air-dried, and allowed to remain at room temperature.

The giemsa stain is used to examine conjunctival specimens for cytology and microorganisms. It could provide pertinent information in about one hour. After a specimen is obtained, the slides are fixed with methanol, dried, and placed in giemsa stain. Under microscopy, a conjunctival specimen from a patient with ocular chlamydia would contain many epithelial cells, many polymorphonuclear cells, few mononuclear cells, and a few plasma cells. This cytology alone could support a chlamydial diagnosis. Chlamydial inclusion bodies (initial, elementary) can also be observed in the cytoplasm of the epithelial cell especially in newborns five to 20 days after birth but less frequently in young and older adults. Although the giemsa stain is a powerful tool, it rarely is offered because of the lack of experienced personnel.

Giemsa stain of Chlamydia inclusion bodies (purple "caps" on epithelial cell).

PCR
Specimens are directly collected by vigorously swiping the exposed conjunctiva with a plastic soft-tipped applicator. Cornea samples are not necessary. Topical anesthetic can be applied to the conjunctiva but this is optional.

Bartels Chlamydia Transport Media
Collected samples are placed in 2.0 ml of chlamydial transport medium. We have had great success with Bartels Chlamydial transport medium and recommend its use. DO NOT USE VIRAL TRANSPORT MEDIUM THAT MAY CONTAIN ANTIBIOTICS THAT INHIBITS CHLAMYDIA GROWTH IN CELL CULTURE. Chlamydia is very fastidious and will not survive unless refrigerated (short-term) or frozen (long-term) (-75°C). All samples for cell culture must be transported immediately to the laboratory. PCR can be processed from the transport medium.

We highly recommend the PCR test for the detection of chlamydial DNA from ocular specimens. This is a 6-hour test that is commercially and widely available. There are no problems with transporting these specimens because live organisms are not necessary and DNA is very stable. Samples should be refrigerated and still transported to the laboratory as soon as possible to avoid any possible DNAses that possibly may degrade any DNA in samples. We advise that ophthalmologists should make the effort to locate PCR testing if they are frequently exposed to patients with a chlamydial differential.

Photo left: PCR reaction; Photo right: Postive Chlamydia PCR tests.