Histopathological diagnosis of Japanese spotted fever using formalin-fixed, paraffin-embedded skin biopsy specimens Usefulness of immunohistochemistry and real-time PCR analysis

Abstract

Japanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology.

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

“…Sequencing of the PCR‐amplified fragments is useful for confirming the bacterial species. In our previous study, genomic DNA fragments of Rickettsia japonica were demonstrated in FFPE sections of skin biopsy of Japanese spotted fever by real‐time PCR and sequencing …”

“…In our previous study, genomic DNA fragments of Rickettsia japonica were demonstrated in FFPE sections of skin biopsy of Japanese spotted fever by real-time PCR and sequencing. 17 We tried herein to detect Ck in FFPE biopsy specimens of GM both by low-specificity immunostaining and by realtime PCR.…”

Bartonella henselae infection caused by cat flea bite

“…Antigens of Rickettsia japonica (a causative agent of JSF) were undetectable by two mouse monoclonal antibodies (clones X1 and S3). 5 A mouse monoclonal antibody to B. henselae (clone: H2A10, Biocare Medical, Concord, CA, USA) diluted at 1:100 identified strongly positive signals in the cytoplasm of macrophages and endothelial cells at the site of ulceration (Fig. 2d).…”

“…Histologically, the eschar was associated with marked lymphoplasmacytic reactions in the deep dermis through subcutaneous tissue, unusual findings in JSF. 5 We have evaluated 50 cases of JSF with skin biopsy specimens for immunohistochemical and PCR demonstration of R. japonica, and both significantly contributed to an early diagnosis of JSF. 5 In our case, the same strategy was effective for making a definite/differential diagnosis of B. henselae infection.…”

“…5 We have evaluated 50 cases of JSF with skin biopsy specimens for immunohistochemical and PCR demonstration of R. japonica, and both significantly contributed to an early diagnosis of JSF. 5 In our case, the same strategy was effective for making a definite/differential diagnosis of B. henselae infection. Skin biopsy trials in making a diagnosis of CSD have been reported.…”