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M Crawford, JA Smith, DA Galita, CC Chan; Ultrastructural Detection of Chlamydia Inclusions in Epithelial Cells Using Conjunctival Swabs . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1600.
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Purpose: Chlamydiceae are a group of intracellular parasites that resemble bacteria in their sensitivity to antibiotics. They are larger than most viruses and commonly cause follicular conjunctivitis. However , the microorganisms can be difficult to diagnose using routine culture. We report identification of chlamydial inclusions in conjunctival epithelial cells obtained from three patients. Methods: Conjunctival epithelial cells were obtained from two patients using Dacron swabs. . One portion was spun down using a Cytopro cytospin for cytology. The second portion was put into an eppendorf tube in 0.5ml PBS and centrifuged. Most of the supernatant was decanted off, and the pellet was fixed in a solution of 2.5% gluteraldehyde in 0.1% cacodylate buffer containing 0.04% Ruthenium Red stain, which makes the pellet more visible. The pellet was processed through ascending alcohols and propylene oxide and embedded in Ladd LX-112 resin. Thin sections were taken, stained with uranyl acetate and lead citrate and observed by Transmission Electron Microscopy, using a Joel 1010 electron microscope . Results: It was challenging to identify both mature and immature inclusion bodies on Giemsa stained cytologic slides. However, chlamydia reticulate and elementary bodies were readily found in the cytoplasm of conjunctival epithelial cells using electron microscopy. The inclusions measured about 0.3 µm and were characterized by dense bodies and membranes of phagocytic vacuoles (phagolysosomes). Some contain binary-like fission reticulate bodies. Subacute inflammatory cells were also present. Conclusion: Examination of conjunctival epithelial cells is a useful and non- invasive procedure for the diagnosis of conjunctival disorders. Evaluation of the conjunctival epithelial cells by transmission electron microscopy is a feasible and informative method for the detection and confirmation of chlamydia infection. This will help patients to promptly receive appropriate antibiotic therapy.
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