PCR can diagnose Acanthamoeba keratitis

DNA testing proved its value after flooding triggered an outbreak in Iowa.

Issue: June 1, 2000

PORTLAND, Ore. — Polymerase chain reaction (PCR) tests are a cheaper and definitive way to diagnose Acanthamoeba keratitis, according to William D. Mathers, MD.

Dr. Mathers saw a tremendous increase in Acanthamoeba infections following floods in Iowa in 1993. Before the floods, he diagnosed about two cases per year. In 1993 and 1994, the infections were frequent and severe. Since then, he has diagnosed about 50 cases annually, although many are milder forms of the disease.

“And we’re not getting it all,” he told Ocular Surgery News. “It could be 100 times more common than is currently appreciated, or more.”

While confocal microscopy is useful and noninvasive, it lacks the resolution needed to make a definitive diagnosis. Culturing results were inadequate, so Dr. Mathers sought a more sensitive method.

“[Acanthamoeba] is hard to diagnose unless you take special steps,” he said. “The most sensitive measure that we have right now that we know of is PCR — looking for the DNA of the organism.”

PCR will be much more widely used in the future than it is now, he said. While mostly used in the lab and not in clinical practice, the technology is becoming more available. The costs are lower than culturing, although clinical practices would need a special setup to conduct PCR tests.

Confirmed results

To confirm the PCR test’s usefulness, researchers used confocal microscopy and histology to diagnose cases, and then confirmed results with PCR.

Researchers reported in the Archives of Ophthalmology that 117 patients entered the University of Iowa’s Cornea Service for treatment of possible Acanthamoeba infection.

Physicians used confocal microscopy to find organisms in the epithelium and anterior stroma. Physicians found suspected organisms in 31 patients and biopsied them. Two patients were found to be negative for Acanthamoeba but underwent biopsy anyway.

The 33 biopsied specimens were divided for histological evaluation and for PCR. Confocal microscopy made a positive diagnosis in 30 cases. Two false-positive patients were negative by microscopy, histology and PCR. One patient was negative by microscopy and histology but positive by PCR.

The two patients with false-positive results may have been Acanthamoeba cases that did not have the correct DNA sequence to work with the three PCR primers Dr. Mathers used.

Sensitive, specific

The small number of Acanthamoeba organisms available from biopsy may be why it is so difficult to diagnose by culturing, Dr. Mathers said.

Researchers wrote that, “Because a single Acanthamoeba organism may contain more than 100 copies of ribosomal DNA, the detection of a very small number of organisms is possible.”

However, they cautioned that the sensitivity of PCR requires care when collecting specimens. And although PCR is more sensitive, it is extremely specific.

Histology also reveals information that PCR does not, such as cell type, severity of inflammation and the appearance of the organisms.

Cultured organisms including both the cyst and the trophozoite forms. The cysts have the double walls.	Culture of trophozoites alone.
Confocal microscopy of Acanthamoeba organisms in the epithelium of a patient with Acanthamoeba keratitis.	Histology of Acanthamoeba.

For Your Information:

William D. Mathers, MD, can be reached at Casey Eye Institute, OHSU, 3375 S.W. Terwilliger Blvd., Portland, OR 97201; (503) 494-3976; fax: (503) 494-3929.

Reference:

Mathers WD, Nelson SE, Lane JL, Wilson ME, Allen RC, Folberg R. Confirmation of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch Ophthalmol. 2000;118:178-183.