Corynebacterium kroppenstedtii in granulomatous mastitis: Analysis of formalin‐fixed, paraffin‐embedded biopsy specimens by immunostaining using low‐specificity bacterial antisera and real‐time polymerase chain reaction

Abstract

Granulomatous mastitis (GM) is a rare inflammatory disease of the post-lactation breast, clinically mimicking breast cancer. GM is microscopically characterized by formation of epithelioid granulomas and abscess (suppurative granulomas) with lipid droplet-centered inflammation. Corynebacterium kroppenstedtii (Ck) is known as a causative bacterium of GM, and identification of Ck infection within the lesion should thus be essential for confirming the diagnosis. In the present study, we analyzed formalin-fixed, paraffin-embedded (FFPE) biopsy specimens of a total of 18 GM lesions with immunostaining and real-time PCR for Ck genome. Widely cross-reactive rabbit antisera against Bacillus Calmette-Guerin (BCG), Bacillus cereus, Treponema pallidum and Escherichia coli were chosen. With real-time PCR, Ck genome was demonstrated in 7 of 18 GM lesions. Immunohistochemically, the low-specificity antisera reacted with the cytoplasm of phagocytes and/or granuloma-engulfed lipid droplets in 12 of 18 GM lesions. Antigenic positivity was observed in the following order: BCG > B. cereus > T. pallidum > E. coli. Real-time PCR using DNA extracted from FFPE sections was useful but not consistent for identifying the Ck genome in GM, while immunostaining using cross-reactive antisera against four kinds of bacteria was not Ck-specific but was applicable to visualizing bacterial infection within the GM lesions.

Pathogens in patients with granulomatous lobular mastitis

“…Using fresh biopsy samples for DNA extraction would undoubtedly improve sensitivity of detection, however, FFPE specimens are more readily available at Shenzhen Traditional Chinese Medicine Hospital. In terms of the detection rate of C. kroppenstedtii, the study results are consistent with those of Fujii et al (35% vs. 38.9%) (Fujii et al, 2018), who detected C. kroppenstedtii in FFPE specimens of granulomatous mastitis using real-time PCR. This indicates that the detection method currently being used at Shenzhen Traditional Chinese Medicine Hospital is reliable.…”

Granulomatous Mastitis: A Therapeutic and Diagnostic Challenge

“…After this finding, further case reports were published presenting different species of corynebacterium (C. kroppenstedii, C. tuberculostearicum, and C. freneyi) [21,22], supporting the theory of Taylor et al [20. ]In a Japanese case series, isolated C. kroppenstedii was found in 6 of 19 cases of GM [23]. Some authors define the finding of C. kroppenstedii within GM lesions as indicative of a subgroup of the disease named ‘cystic neutrophilic granulomatous mastitis' because of its histopathologic presentation of granulomatous and neutrophilic inflammation with cystic spaces or vacuoles [7].…”

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens in Formalin-Fixed, Paraffin-Embedded Sections

“…Immunoperoxidase application using four kinds of commercially available rabbit antisera raised against BCG, B. cereus, T. pallidum, and E. coli is described [4,7,8,16,19]. In this section, immunostaining application to mycobacterial infection, B. cereus pneumonia, syphilitic lesions, and E. coli infections is described.…”

“…We have a limited number of commercially available antibodies against pathogens. Useful commercial antibodies may soon disappear from the market because of a simple reason: the dead stock [16].…”

“…In order to visualize pathogens in formalin-fixed, paraffin-embedded sections, we do not necessarily need to prepare antibodies with high specificity [4,7,19]. The author routinely uses four kinds of commercially available rabbit antisera raised against Bacillus Calmette-Guérin (BCG; Mycobacterium bovis), Bacillus cereus (B. cereus), Treponema pallidum (T. pallidum), and Escherichia coli (E. coli) [16,19]. For immunoperoxidase staining, the amino acid polymer technique or indirect immunoperoxidase method is employed.…”