High-frequency ultrasound biomicroscopy and multifrequency ultrasound

High-frequency ultrasound biomicroscopy (UBM) allows detailed imaging of the anterior segment of the eye, ciliary body, pars plana, and peripheral vitreous. Because many patients with uveitis have severe media opacity that impairs the clinician’s ability to examine these structures clinically, UBM can allow assessment of inflammatory lesions not otherwise visible. In our experience, UBM is useful in selected patients with uveitis. UBM also can help in assessing inflammation in patients with severe media opacity, in assessing the placement of an intraocular lens to see whether it may be contributing to inflammation, and in evaluating causes of hypotony because it can provide detailed information on the anatomy of the ciliary body. Recently, Tran and colleagues58 retrospectively reviewed UBM findings from 111 eyes in 77 patients with uveitis. They noted that UBM findings contributed to the diagnosis or had an impact on treatment in 43% of patients. In a study of seven eyes in five patients with intermediate uveitis, ultrasound examination with both 50and 20-MHz frequency probes were able to detect snowbanks and may be useful in eyes with small pupils or dense vitritis to assess disease activity.59

Fundus autofluorescence

Liebman and Leigh described autofluorescence of visual receptors in an aritcle in Nature in 1969.60 More recently, autofluorescence has been used to assess pathologic changes to the retinal pigment epithelium that are thought to be associated with clinically relevant changes in function. Autofluorescence may be useful in assessing changes to the RPE that may be missed with other imaging modalities. The

technique has been used to assess patients with a number of retinal conditions, including age-related macular degeneration and uveitis. For example, focal accumulation of auto­ fluorescent material was shown at the level of the RPE in patients with central serous chorioretinopathy.61 Autofluorescence was also used to show more widespread involvement of the RPE than previously documented in patients with multifocal choroiditis and panuveitis.62

Other diagnostic tests

Polymerase chain reaction (PCR)

Since its initial description by Saiki and colleagues,63 the polymerase chain reaction (PCR) has become one of the most commonly used techniques in biomedical research. It provides a simple way to amplify a specific fragment of DNA, and this technique has proved useful for the study of genetic diseases and the detection of infectious agents. Pertinent to the field of uveitis, PCR has also been used to detect viruses, bacteria, and parasites in the eye.64

To start the PCR four components are combined: (1) target DNA, (2) primers (short strands of DNA that tag the section of target DNA to be copied, (3) polymerase (the enzyme that catalyzes gene replication), and (4) nucleotides. The PCR is based on the consecutive repetition of three reactions. First, the target DNA is denatured by heating the test tube to 95°C. Second, primers are annealed to a targeted strip of DNA as the temperature of the test tube is reduced. Finally, in the third step of the PCR polymerase triggers the synthesis of a new DNA molecule between the primers. These three steps of the reaction are then repeated

Box 5-4  FDA-Approved PCR tests for infectious

agents associated with uveitis

HIV-1

Hepatitis C virus

Mycobacterium tuberculosis

Neiserria gonorrhea

Chlamydia trachomatis

Aspergillus galactomannan

consecutively during 30–40 cycles and controlled by the raising and lowering of the temperature of the test tube.

At the end of the PCR, the amount of target DNA is increased by 1 millionto 1 billion times. However, the extreme sensitivity of the technique requires very strict laboratory procedures to prevent carryover of positive substrate between samples that could lead to false-positive results. The PCR can be used to detect DNA fragments in many types of specimens including cells, body fluids, and paraffinembedded sections. The PCR is currently being used to identify the DNA of infectious agents in the eye, because, as we have seen, the interpretation of serologic tests is often misleading. Before PCR can be carried out, the sequence of the target DNA (specific for the organisms in question) must be available, and the appropriate primers must then be syn­ thesized. Viral DNA has been identified by PCR in eyes with a number of disorders including acute retinal necrosis.65 We currently use the PCR in our laboratory to diagnose ocular toxoplasmosis.66,67

The US Food and Drug Administration has approved nucleic acid amplification tests for the detection of a number of infectious agents that can also cause uveitis (Box 5-4). PCR tests are now commercialy available for Mycobacterium tuberculosis.68 Initially, these tests were only indicated for use in patients with respiratory specimens that produced negative results on acid-fast bacilli smears. These tests are now thought to be a reasonable adjunctive test in all patients with presumed tuberculosis. Of course, these tests should not replace the standard acid-fast bacilli smear or mycobacterial cultures, and should only be interpreted in the context of other clinical information. Unfortunately, these commercial tests have not been well studied for the diagnosis of tuberculosis from ocular specimens, although there are studies showing that PCR can detect Mycobacterium tuberculosis from the vitreous in some patients.69,70 There are now commercially available PCR tests for other infectious agents that can cause uveitis. FDA-approved tests to detect Niesseria gonorrheae, Chlamydia trachomatis, HIV, hepatitis C virus, and

Aspergillus can now be ordered. Importantly, noncommercial PCR testing can be useful in elucidating infectious causes of uveitis. Tropheryma whipplei was identified by PCR from the vitreous of a patient with uveitis associated with Whipple’s disease.71 To date, PCR is still predominantly a research tool. Because of its exquisite sensitivity, the chances of false-pos- itive results are significant. In addition, a small piece of DNA found in the eye of a patient may represent a remnant from an infection that occurred long ago and may be totally unrelated to the patient’s current illness. Nevertheless, the use of PCR will continue to proliferate in ophthalmic research, and it will probably be available clinically in the near future to detect infectious agents in ocular specimens.

Other diagnostic tests

Rapid tests for herpes simplex and herpes zoster

Currently, biopsy specimens, including cells obtained from anterior chamber taps, are tested for herpesvirus with the use of a direct fluorescent antibody test after 48 hours and 5 days of growth. Enzyme-linked immunosorbent assays, which give almost immediate diagnostic information, are available. However, problems with sensitivity and specificity arise and need to be thoroughly investigated before these tests can be used routinely in clinical practice.

Bone mineral density studies

Historically, clinical tests including laboratories tests and imaging studies were performed to make a diagnosis or to assess the efficacy of therapy. Diagnostic tests are now used to monitor treatment toxicity. As we have stated, cortico­ steroids remain the mainstay of therapy for uveitis. Unfortunately, researchers have shown that corticosteroids can promote osteoporosis. One study showed that patients receiving prednisone at a dose greater than 7.5 mg/day lost 10–15% of trabecular bone in the lumbar spine within 1 year.72 At prednisone doses greater than 30 mg/day bone loss increased to 30–50%. In one study of corticosteroid-induced osteoporosis in patients with uveitis, seven symptomatic fractures occurred in 129 patients during treatment.73 Bone mineral density studies can accurately assess bone loss in patients treated with corticosteroids. If there is a reasonable chance for chronic steroid use, bone mineral density studies should be performed within 3 months of the start of systemic corticosteroid therapy and annually thereafter. If these studies show osteoporosis, patients may benefit from anti­ resorptive agents such as calcitonin, alendronate, etidronate, or risedronate.

Genetic testing for steroid-induced glaucoma

Glaucoma is a major cause of visual loss in patients with uveitis. Glaucoma occurs in many patients with uveitis in the presence of active inflammation. In addition, glaucoma can occur as a side effect of corticosteroid use. To date, it has not been possible to predict which patients with uveitis are at greatest risk for glaucoma. Stone and coworkers74 analyzed sequence-tagged site content and haplotype sharing between families affected with chromosome 1q-linked open-angle glaucoma to prioritize candidate genes for mutation screening. These researchers identified a gene encoding a trabecular meshwork protein (TIGR) associated with glaucoma. Polansky and associates75 showed that glucocorticoids and oxidative stress stimuli induced TIGR in cultures of human trabecular meshwork cells, suggesting a mechanism of action for both corticosteroid-induced glaucoma and uveitic glaucoma. Clinical testing for genes associated with glaucoma, such as TIGR, may help clinicians predict which patients are at greatest risk for developing glaucoma, but to date is not routinely used in clinical practice.

Neurologic tests

The eye is a part of the brain, and many forms of uveitis have associated central nervous system involvement. Because the

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