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Propionibacterium acnes endophthalmitis diagnosed by microdissection and PCR
  1. R R Buggage1,
  2. D F Shen1,
  3. C-C Chan1,
  4. D G Callanan2
  1. 1Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD, USA
  2. 2Texas Retina Associates, Arlington, TX, USA
  1. Correspondence to: Ronald R Buggage, MD, NIH/NEI, Building 10, Room 10N112, Bethesda, MD 20892-1857, USA; buggager{at}nei.nih.gov

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Although Propionibacterium acnes, a Gram positive anaerobic bacillus, is the most commonly identified cause of delayed onset postoperative endophthalmitis, routine vitreous cultures are frequently inadequate for its diagnosis. This case describes the utility of the histopathological technique of microdissection and polymerase chain reaction (PCR) for the diagnosis of delayed postoperative endophthalmitis.

Case report

A 78 year old man with a history of vitreous floaters, a coronary bypass, and aortic valve replacement underwent an uncomplicated cataract extraction with intraocular lens (IOL) implantation in the right eye. Three months later, he developed increasing floaters in the right eye and was diagnosed with vitritis unresponsive to corticosteroid treatment. Examination revealed acuities of 20/25 in the right eye and 20/20 in the left with normal intraocular pressures. The right eye was significant for no anterior chamber cells or flare, dilated iris vessels, an IOL without deposits, 3+ vitreous cells with trace haze, and peripheral pigmentary degeneration. The left eye was normal with the exception of trace vitreous cells and a choroidal naevus. A diagnostic vitrectomy was performed in the right eye. A portion of the vitreous specimen was cultured for fungi, aerobic and anaerobic bacteria, and the remainder was processed for cytopathological examination. All cultures for micro-organisms were negative.

The vitreous supernatant and unstained cytology slides were sent to the National Eye Institute for further evaluation. Vitreal analysis for interleukin 2 (IL-2), IL-4, IL-6, IL-10, IFN-γ, and TNF-α using ELISA (Endogen, Woburn, MA, USA) revealed undetectable cytokine levels. The vitreous slides were stained with Giemsa, Gram, and immunohistochemical stains for T cells, B cells, and macrophages. Cytopathological examination showed clusters of macrophages admixed with CD4+ and CD8+ T cells and B cells (Fig 1A). Gram positive bacilli were seen in the cytoplasm of a few macrophages (Fig 1B). The engulfed bacilli were microdissected under a microscope with a 30 gauge needle and submitted for PCR.1 Nested PCR with P acnes specific oligodeoxynucleotide primers complementary to regions of 16S rDNA was used.2 The primers were Pa1, AAG GCC CTG CTT TTG TGG; rPa2, TCC ATC CGC AAC CGC CGA A; and rPa3, ACT CAC GCT TCG TCA CAG. Nested-PCR analysis revealed P acnes (Fig 2). A diagnosis of delayed postoperative endophthalmitis was made.

Figure 1

(A) Photomicrograph of the vitreous specimen showing clusters of macrophages admixed with T and B lymphocytes. Many degenerated lymphocytes were also present. (Giemsa, ×200). (B) Higher power photomicrograph of the vitreous specimen showing Gram positive pleomorphic bacilli (arrow) in the cytoplasm of a few macrophages (Gram stain, ×640).

Figure 2

Nested PCR products of the microdissected bacilli from the vitreous specimen. The first round used Pa1 and rPa2. The second round used Pa1 and rPa3. The negative control in both rounds contained no DNA. The positive control in both rounds was P acnes.

Comment

The most common causes of vitritis in elderly patients are acquired or postoperative infections, sarcoidosis, and intraocular malignancies masquerading as uveitis.3 An early diagnostic procedure is indicated if postoperative endophthalmitis is suspected. In this case, although the chronic inflammation and intracytoplasmic Gram positive bacilli in a few macrophages suggested an infectious process, the negative cultures precluded the diagnosis of an infectious endophthalmitis. To further investigate the possibility of a bacterial infection nested PCR was performed on the microdissected bacilli.1 Molecular analysis verified the presence of P acnes and a diagnosis of delayed postoperative endophthalmitis was confirmed.

Vitreous cultures are positive in less than 50% of postoperative endophthalmitis cases. In a study of 25 patients with delayed onset endophthalmitis aqueous culture and microscopy were diagnostic in 0% of cases, vitreous culture was positive in 24% and PCR from the aqueous and vitreous yielded a positive diagnosis in 84% and 92%, respectively.4 Treatment of P acnes endophthalmitis includes intravitreal vancomycin plus consideration of pars plana vitrectomy with or without capsulectomy with or without IOL removal. Although aggressive surgical intervention eradicates the infection similar visual outcomes are reported with more limited surgical treatment.5

In our case the intracytoplasmic bacteria in the macrophages were the only evidence of a bacterial infection. To detect the presence of P acnes we referenced the PCR method described by Hykin that used 150 μl of the vitreous for culture and 100 μl for PCR.2 Using the technique of microdissection and PCR with a similar volume of vitreous we additionally performed cytology and cytokine analysis which are helpful in the diagnosis of other causes of vitritis.6

This case further illustrates the benefits of molecular analysis for the diagnosis of culture negative delayed onset endophthalmitis. It also describes for the first time microdissection and PCR for the evaluation of endophthalmitis. Advantages of this technique are that it allows for a more comprehensive pathological examination on a limited specimen and provides the option of having the molecular studies being performed elsewhere.

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