Photo Quiz: PAS- and GMS-positive filamentous branching organisms in an immunocompromised patient

 PHOTO QUIZ

A 49-year-old female presented to the Dermato-oncology Clinic of Stanford Health Care with a newly developed, painless, 1-cm non-tender violaceous nodule on her left breast. Her past medical history was significant for acute myeloid leukemia. She was status post two cycles of chemotherapy with daunorubicin and cytarabine followed by an allogeneic stem cell transplant 6 months prior, and her latest bone marrow biopsy showed complete remission. Her transplant was complicated by Grade III/IV gastrointestinal graft-versus-host disease, for which she was currently receiving treatment with daily high-dose steroids. The patient did not report any recent history of travel or residence outside of California, nor any unusual zoonotic or occupational exposures.

A 4-mm punch biopsy was performed and sent to pathology for histopathologic examination. No bacterial, mycobacterial, or fungal cultures were sent for microbiological testing. Histopathological examination revealed a large, dermal neutrophilic abscess (Fig. 1A). Grocott’s methenamine silver (GMS) and periodic acid Schiff (PAS)’s stains were positive for numerous thin (0.6–0.8 μm), medium length, filamentous branching structures, while Fite staining, which uses a strong acid during the decolorizing step, was negative for acid-fast bacilli (Fig. 1B through D). Gram stain demonstrated these structures to be variably gram-positive with a beaded staining pattern (Fig. 1E). Given the presence of organism on the histopathology, the microbiology medical director and pathology trainees on service were consulted by the dermatopathology department for further morphologic assessment and recommendations for molecular testing.

Fig 1.

Histopathological examination of the cutaneous breast nodule. Hematoxylin and eosin stain, 100× (A). Grocott’s methenamine silver stain, 100× (B). Periodic acid Schiff stain, 100× (C). Fite stain, 100× (D). Gram stain, 100× (E). Panels B, C, and E reveal numerous thin, filamentous branching structures (arrows), which were positive for Grocott’s methenamine silver (GMS) and periodic acid Schiff (PAS) stains and variable on gram stain.

Incidentally, the patient had also been complaining of a cough, and later workup included a CT chest that demonstrated a dense consolidation of the left lower lobe suggestive of pneumonia, in addition to scattered bilateral pulmonary nodules. Additional imaging of her abdomen and pelvis did not reveal any infectious foci. A review of symptoms was negative for central nervous system symptoms, including headaches or mental status changes. A CT head was performed, which showed no intracranial abnormalities or lesions.

What is your diagnosis?

ANSWER TO PHOTO QUIZ

The patient was diagnosed with Nocardia farcinica complex. The morphologic appearance raised a microbiological differential diagnosis that predominantly included Nocardia, Actinomyces, and Streptomyces as well as molds given the positive GMS and PAS staining. Bacterial 16S rRNA gene amplicon sequencing of the formalin-fixed paraffin-embedded (FFPE) tissue was performed (GenBank: OR684569.1), identifying Nocardia farcinica complex. The patient was diagnosed with a disseminated nocardiosis infection and was started on trimethoprim–sulfamethoxazole with clinical improvement of symptoms.

Given their filamentous and branched morphology, Nocardia spp. can be mistaken for fungal elements; however, these organisms are much thinner (0.5–1 μm in diameter) compared to true fungal hyphae (usually 2–10 μm in diameter), do not have the presence of septations, and exhibit a beaded staining pattern on gram stain (1–5). GMS and PAS are stains typically used to identify the presence of fungi during histopathologic evaluation, as these stains react with polysaccharides and carbohydrate containing macromolecules found in the fungal cell wall, respectively. Although the organisms in this case were noted to be GMS-positive, all gram-positive bacteria, including the Actinomycetes, stain with GMS (2); however, the positive PAS staining was somewhat puzzling as Nocardia spp. are typically described as being PAS-negative (2, 3, 6, 7); however, rare cases in the literature have reported this phenomenon (8–11). Nocardia stains positively with modified acid-fast stain and modified Fite stain, which both use weak acid in the decolorizing step. Unlike the Fite stain, which was negative in this case, the modified acid-fast stains can be helpful in identifying Nocardia on histopathology.

This case highlights the challenges in making a histopathologic distinction between branching, filamentous bacteria, and fungal hyphal elements, reinforcing the importance of sending the appropriate microbiologic testing when an infectious diagnosis is suspected, especially in immunocompromised hosts. Fortunately, the availability of broad-range sequencing on FFPE tissue helped to identify the causative agent, which would have otherwise been difficult to identify by histopathologic findings alone. Although the presence of PAS positivity usually indicates the presence of fungal organisms, filamentous bacteria, such as Nocardia spp., must be taken into consideration as an etiology, particularly when the PAS-positive structures are thin (<2 µm in diameter).