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Clinicopathologic Reports, Case Reports, and Small Case Series
February 2004

Diagnosis of Microsporidia Keratitis by Polymerase Chain Reaction

Arch Ophthalmol. 2004;122(2):283-284. doi:10.1001/archopht.122.2.283
Report of a Case.

A 33-year-old man positive for human immunodeficiency virus had a 6-monthhistory of bilateral blurred vision, tearing, and photophobia, with associatedconjunctival hyperemia. The patient had been previously treated with topicalantibiotic drops, topical steroids, and topical nonsteroidal anti-inflammatorydrops for several months without resolution of his symptoms. He was takinghighly active antiretroviral therapy as well as trimethoprim-sulfamethoxazoleprophylaxis; his CD4 cell count was 3. His medical history included weightloss and diarrhea. His ocular history was otherwise unremarkable. His uncorrectedvisual acuity was 20/70 OD and 20/50 OS with pinhole to 20/50 OD and 20/30OS. The conjunctiva had 2+ hyperemia with a moderate papillary reaction bilaterally.The corneas exhibited diffusely distributed small, gray intraepithelial lesionswithout associated stromal infiltrate (Figure1). There was no anterior chamber reaction, and the posterior segmentswere unremarkable.

Figure 1.
Intraepithelial keratitis in ahuman immunodeficiency virus–positive patient.

Intraepithelial keratitis in ahuman immunodeficiency virus–positive patient.

The patient underwent a conjunctival biopsy and cytologic smear examination.Gram stain, bacterial culture, fungal culture, chlamydial testing, and viralculture were negative. Cytologic examination was negative for microsporidiaor typical viral or chlamydial inclusion bodies. A fine-needle corneal epithelialscraping was performed. The scraped sample was suspended in 50 µL ofphosphate-buffered saline and boiled for 15 minutes. Polymerase chain reaction(PCR) for microsporidia was performed using a protocol adapted from Mulleret al,1 which is capable of identifyingseveral Enterocytozoon and Encephalitozoon species of microsporidia. Briefly, 5 µL of the sample was subjectedto 35 cycles of thermocycling using forward primer V1 (5′-CACCAGGTTGATTCTGCCTGAC-3′)and reverse primer PMP2 (5′-CCTCTCCGGAACCAAACCCTG-3′) with 1-minutedenaturation at 94°C, 2-minute annealing at 60°C, and 3-minute extensionat 72°C. A single ∼270–base pair fragment was observed on agarosegel electrophoresis and ethidium bromide staining of the PCR-amplified patientsample, but not from the phosphate-buffered saline–only control sample(Figure 2A). The PCR product wasdirectly sequenced. A BLAST search of the National Center for BiotechnologyInformation database revealed a near-perfect alignment with the ribosomalRNA small unit gene of Encephalitozoon hellum (Figure 2B). The patient was prescribed hourly1% topical clotrimazole and 100 mg of oral itraconozole 2 times daily, andshowed gradual improvement in his symptoms and clinical findings during a2-week period.

Figure 2.
A, Polymerase chain reaction (PCR)for microsporidia. B, Alignment of the sequence of PCR product (bottom line)with a ribosomal DNA gene from Encephalitozoon hellum (topline).

A, Polymerase chain reaction (PCR)for microsporidia. B, Alignment of the sequence of PCR product (bottom line)with a ribosomal DNA gene from Encephalitozoon hellum (topline).


Microsporidia are a group of at least 6 genera of intracellular protozoathat are frequent opportunistic pathogens in immunocompromised patients, particularlyinfecting the gut. Diagnosis is challenging and has generally relied on directvisualization of the parasites on cytologic or histologic sections.2 Microsporidia keratitis has been reported in immunocompromisedhosts3 but has been difficult to diagnose;the sensitivity of direct observation for detection of the pathogen is unknown.Although serologic testing is positive in patients with microsporidia keratitis,the specificity of this finding in patients with human immunodeficiency virusis also unknown.4 Our report demonstratesthat the PCR, which is useful in the diagnosis of a number of ocular infectiousconditions,5 may be successfully appliedto the diagnosis of microsporidia keratitis and may have higher sensitivitythan traditional cytologic and histologic detection methods. Its specificityis presently unknown. Further studies to determine the sensitivity, specificity,and positive and negative predictive values of PCR for microsporidia keratitiswill define the full clinical utility of this test.

Dr Van Gelder is supported by the Becker/Association of University Professorsof Ophthalmology/Research to Prevent Blindness Physician-Scientist Award,the Culpepper Medical Scientist Award of the Rockefeller Brothers Foundation,and a career development award from Research to Prevent Blindness Inc, NewYork, NY.

The authors have no relevant financial interest in this article.

This study was performed in accordance with Washington University MedicalSchool (St Louis, Mo) institutional review board protocol.

Corresponding author: Russell N. Van Gelder, MD, PhD, Departmentof Ophthalmology and Visual Sciences, Washington University Medical School,Campus Box 8096, 660 S Euclid Ave, St Louis, MO 63110 (e-mail: vangelder@vision.wustl.edu).

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