Nonspecific Granular Reactivity of EBER In-Situ Hybridization in a Patient of Black Hairy Tongue

To the Editor: Epstein–Barr virus (EBV) is well known to be associated with various mucocutaneous disorders in immunosuppressed hosts, especially post-transplant patients. Some of these are oral hairy leukoplakia, EBV-positive mucocutaneous ulcers, and EBV-related cutaneous lymphoproliferative disorders.1 The method of choice for detecting EBV infection in formalin-fixed tissue remains in situ hybridization for EBV-encoded RNA (EBER).2 Positive EBER in situ hybridization (EBER ISH) test presents as blue nuclear staining of the latently infected cells in formalin-fixed tissue.3,4 Cross-reactivity or false staining of the test has been reported with yeasts, plants, and mucin.5,6 Cross-reactivity with EBER in subcutaneous aluminum granulomas at the site of prior vaccination, presenting as cytoplasmic staining in aluminum-containing histiocytes, has also been documented.7 The mechanism of these cross-reactivities has not yet been confirmed. We report a 70-year-old man, status of 10 years post liver transplant for hepatocellular carcinoma and receiving tacrolimus. He had a history of multiple cutaneous squamous cell carcinomas. In his latest surgery to remove a cutaneous squamous cell carcinoma, the surgeon also biopsied a tongue lesion that was described as a firm lesion on the dorsal tongue. Biopsy showed acanthotic squamous mucosa with bacterial colonies in the superficial keratotic layers consistent with lingua nigra (black hairy tongue) without evidence of dysplasia or malignancy. Owing to his medical history and the location of the lesion, EBER ISH test was performed to exclude EBV effect. With appropriate positive and negative controls, the keratinocyte nuclei were negative. However, there was a fine granular positivity on the surface of the keratinous layer in the area that we could see superficial bacterial colonies on the H&E-stained slide (Fig. 1).FIGURE 1.: A and B, Hematoxylin and eosin (H&E)-stained low- and high-power views of the tongue biopsy specimen, showing hyperkeratosis and acanthosis with bacterial colonies present in the superficial keratotic layer. No evidence of dysplasia or malignancy is observed. C, Epstein–Barr virus-encoded RNA in situ hybridization (EBER-ISH), high-power view, demonstrating fine granular positivity superficial to the keratotic layer.In patients of black hairy tongue, the surface is usually covered by a mixture of debris (including keratin and food, such as pigment-containing drinks like tea or coffee) and bacteria. Whether the fine granular staining seen in our patient is solely due to the stained bacteria or nonspecific absorption of the surface mixture of debris and bacteria cannot be definitively determined. To the best of our knowledge, cross-reactivity of EBER ISH with bacteria has not been previously reported and the possibility of EBV infection of the bacteria is likely biologically implausible, because EBV primarily infects human B cells and epithelial cells. Regardless of whether bacteria or the mixture of debris and bacteria on the surface show cross-reactivity, this represents a false-positive reaction. This observation further supports the idea that proper interpretation of ancillary tests requires evaluation of the H&E histologic context to avoid possible pitfalls.

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