Transmission electron microscopic study of the surface layer of surgical resected disc specimens in human temporomandibular joint.

In this study, we investigated specific and characteristic findings of the surface layer of surgical resected disc specimens in human temporomandibular joint osteoarthritis cases by transmission electron microscopy (TEM).Specimens were surgically removed from the TMJ of 5 cases (4 female patients: 5 cases) clinically osteoarthritis. Following findings were observed by TEM. Images were photographed on a JEM1400-Flash Electron microscope (JEOL, Japan) equipped with an EM-14661FLASH high-sensitivity digital complementary metal-oxide-semiconductor camera.Following findings were observed by TEM. 1) The surface is covered with plump fibroblastic and histiocytoid cells. 2) Collagen fiber bundles and collagenous matrix are exposed onto the eroded disc surface. 3) Fibrinous dense material is observed on the eroded disc surface. 4) Bundles of collagen fibers are densely observed. 5) Collagen bundles are rich around capillary vessels. 6) Synovial surface cells reveal features of activated macrophages with vacuole formation. Especially, plump fibroblastic and histiocytoid cells, and activated macrophages with vacuole, which were significant findings of the surface layer. These findings might have a significant effect on the regulation of synovial fluid.

[Successful treatment with tucidinostat in a patient with relapsed adult T-cell leukemia/lymphoma following allogeneic hematopoietic stem cell transplantation].

Aggressive types of adult T-cell leukemia-lymphoma (ATL), namely, the acute type, lymphoma type, and chronic type with poor prognostic factors, have a poor prognosis. Although allogeneic hematopoietic stem cell transplantation (HSCT) may improve prognosis, relapse is common. In June 2021, tucidinostat was approved for relapsed or refractory ATL in Japan. We report a case of a 62-year-old man with relapsed ATL after allogeneic HSCT. In March 2017, he was diagnosed with ATL (acute type) and received two courses of mLSG-15 therapy. ATL cells reappeared in his peripheral blood, so he underwent allogeneic bone marrow transplantation in September 2017. In June 2021, his soluble interleukin-2 receptor (sIL-2R) level increased, and he began experiencing sensory abnormalities in his face and legs. In September, he developed respiratory failure and was diagnosed with relapse of ATL. He was again treated with mLSG-15. His sIL-2R normalized and the sensory abnormalities decreased, but sIL-2R rose again in February 2022. After tucidinostat treatment was initiated, sIL-2R normalized and the patient's general condition improved. Tucidinostat shows promise as an effective treatment for ATL that has relapsed after allogeneic HSCT.

Subclinical minute FLT3-ITD clone can be detected in clinically FLT3-ITD-negative acute myeloid leukaemia at diagnosis.

Recent advances in next-generation sequencing (NGS) have enabled the detection of subclinical minute FLT3-ITD. We selected 74 newly diagnosed, cytogenetically normal acute myeloid leukaemia (AML) samples in which FLT3-ITD was not detected by gel electrophoresis. We sequenced them using NGS and found minute FLT3-ITDs in 19 cases. We compared cases with clinically relevant FLT3-ITD (n = 37), cases with minute FLT3-ITD (n = 19) and cases without detectable FLT3-ITD (n = 55). Molecular characteristics (location and length) of minute FLT3-ITD were similar to those of clinically relevant FLT3-ITD. Survival of cases with minute FLT3-ITD was similar to that of cases without detectable FLT3-ITD, whereas the relapse rate within 1 year after onset was significantly higher in cases with minute FLT3-ITD. We followed 18 relapsed samples of cases with clinically FLT3-ITD-negative at diagnosis. Two of 3 cases with minute FLT3-ITD relapsed with progression to clinically relevant FLT3-ITD. Two of 15 cases in which FLT3-ITD was not detected by NGS relapsed with the emergence of minute FLT3-ITD, and one of them showed progression to clinically relevant FLT3-ITD at the second relapse. We revealed the clonal dynamics of subclinical minute FLT3-ITD in clinically FLT3-ITD-negative AML. Minute FLT3-ITD at the initial AML can expand to become a dominant clone at relapse.

Dominant-negative type of IKZF1 deletion showed a favorable prognosis in adult B-cell acute lymphoblastic leukemia.

IKZF1 deletion is a recurrent genomic alteration in B-cell acute lymphoblastic leukemia (B-ALL) and is divided into dominant-negative (DN) and loss of function (LOF) deletions. The prognostic impact of each deletion has not been fully elucidated. We retrospectively analyzed 117 patients with adult B-ALL including 60 patients with BCR::ABL1-positive B-ALL and 57 patients with BCR::ABL1-negative B-ALL by the fluorescence in situ hybridization (FISH) method for IKZF1 deletion and multiplex PCR for the 4 most common IKZF1 deletions (∆4-7, ∆2-7, ∆2-8, and ∆4-8). Samples, in which IKZF1 deletion was detected by FISH but a specific type of deletion was not identified by the PCR, were categorized as "other." Patients were classified into a DN group that had at least 1 allele of ∆4-7 (n = 23), LOF and other group (n = 40), and wildtype group (n = 54). DN type IKZF1 deletions were found in 33.3% of BCR::ABL1-positive cases and 5.2% of BCR::ABL1-negative cases. LOF and other type IKZF1 deletions were found in 43.4% of BCR::ABL1-positive cases and 24.6% of BCR::ABL1-negative cases. Patients with the DN group showed significantly higher overall survival (OS) than that of the LOF and other and WT groups (P = 0.011). Multivariate analysis including age, WBC counts, complex karyotype, and DN type IKZF1 deletion showed that the DN type of IKZF1 deletion (HR = 0.22, P = 0.013) had a positive impact and age ≥ 65 (HR = 1.92, P = 0.029) had a negative impact on OS. The prognostic impact of IKZF1 deletion depends on the type of deletion and DN type of IKZF1 deletion showed better prognosis in adult B-ALL patients.Clinical trial registration This study was part of a prospective observational study (Hokkaido Leukemia Net, UMIN000048611). It was conducted in compliance with ethical principles based on the Helsinki Declaration and was approved by the institutional review board of Hokkaido University Hospital (#015-0344).

Periprosthetic joint infection due to Mycoplasma hominis in a multiple sclerosis patient treated with fingolimod.

Mycoplasma hominis is a commensal pathogen normally found in urogenital tract of humans and has been associated with a wide variety of extra-genitourinary infections, such as mediastinitis, bacteremia, and septic arthritis, particularly in immunocompromised patients. Here, we present a case of a 48-year-old male, who had been treated with fingolimod for relapsing multiple sclerosis and presented with fever and right-sided hip pain following total hip arthroplasty. CT scan revealed localized fluid collection in the right quadriceps femoris muscle adjacent to the joint cavity of right hip. The percutaneously aspirated fluid grew M. hominis, which was also isolated from blood culture. With diagnosis of periprosthetic joint infection, the patient underwent surgical debridement with retained prosthesis and was treated with antimicrobial agents. Infected granulation tissues excised from the hip was observed under an electron microscope, which revealed electron-dense rounded structures contained in neutrophils, consistent with Mycoplasma particles. Fingolimod, an immunomodulatory drug that acts on the sphingosine-1-phosphate receptor and prevents the egress of lymphocytes from lymph nodes, might increase host susceptibility to a systemic M. hominis infection.

Prospective Phase 2 Study of Umbilical Cord Blood Transplantation in Adult Acute Leukemia and Myelodysplastic Syndrome.

Almost comparable transplantation outcomes have been reported with HLA-matched unrelated donor transplantation (UDT) and cord blood transplantation (CBT). We conducted a prospective phase 2 study to assess the efficacy and safety of single-unit myeloablative CBT in adult leukemia and myelodysplastic syndrome. Because the day 180 survival of UDT was approximately 80%, we determined the alternative hypothesis of expected day 180 survival with a successful engraftment rate of 80% and set the null hypothesis of threshold rate at 65%. Sixty-two patients (median age, 37 years) were registered, including 28 with acute myelogenous leukemia, 25 with acute lymphoblastic leukemia, and 9 with myelodysplastic syndrome. Of 61 eligible patients, 52 were successfully engrafted and survived at day 180 (85%; 95% confidence interval, 74% to 93%). Single-unit CBT was judged to be effective because the null hypothesis was rejected (P < .001). Furthermore, neutrophil engraftment was observed in 57 patients (92%); the incidences of grade II-IV acute and chronic graft-versus-host disease were 30% and 32%, respectively; and the cumulative incidences of nonrelapse mortality and relapse at 2 years were 18% and 13%, respectively. The present study showed favorable survival outcomes with single-unit CBT. Therefore, this method may be considered if a well-HLA-matched UDT cannot be obtained.

Colitis nucleomigrans: The third type of microscopic colitis (part 2). An ultrastructural study.

In the preceding article (part 1), we proposed the third type of microscopic colitis: colitis nucleomigrans (CN). Microscopically, the nuclei of surface-lining columnar cells were migrated in chain to the middle part of the cells, and apoptotic nuclear debris was scattered in the cytoplasm beneath the nuclei. For ultrastructural analysis, buffered formalin-fixed biopsy tissue of CN (n = 2) was dug out of paraffin blocks. After deparaffinization, tissue blocks were prepared with conventional sequences. Ultrathin sections were stained with uranyl acetate and lead citrate. Fine morphological preservation was satisfactory even after paraffin embedding. Apoptotic nuclear debris was localized within the cytoplasm beneath the migrated nuclei of the surface-lining columnar cells. Abnormality of cytoskeletal filaments (actin, cytokeratin and tubulin) was scarcely recognized in the epithelial cytoplasm. Macrophages located in the uppermost part of the lamina propria phagocytized electron-dense globular materials. Intraepithelial lymphocytes with scattered dense bodies were observed among the columnar cells. We suppose that altered apoptotic processes in the colorectal surface-lining epithelial cells may be involved in the pathogenesis of CN. Mechanisms of nuclear migration to the unusual position or impairment of nuclear anchoring to the basal situation in the surface-lining epithelial cells remain unsettled, because cytoskeletal components showed little ultrastructural abnormality.

Colitis nucleomigrans: The third type of microscopic colitis (part 1).

Microscopic colitis (MC), encompassing collagenous colitis and lymphocytic colitis, is featured by chronic diarrhea, normal-looking endoscopic findings and unique microscopic appearance. After reviewing biopsied nonspecific colitis, we propose the third type of MC: colitis nucleomigrans (CN). Histopathological criteria of CN included: (i) chained nuclear migration to the middle part of the surface-lining columnar epithelium; (ii) apoptotic nuclear debris scattered below the nuclei; and (iii) mild/moderate chronic inflammation in the lamina propria. Thirty-three patients (M:F = 20:13; median age 63 years, range 17-88) fulfilled our criteria. Seven cases demonstrated MC-like clinical/endoscopic features. Mucosal reddening with or without erosion/aphtha was endoscopically observed in the remaining 26 cases with inflammatory bowel disease (IBD)-like features: occult/gross hematochezia seen in 19, abdominal pain in two and mucin secretion in two. Cleaved caspase-3-immunoreactive apoptotic debris appeared more frequently in IBD-like CN than in MC-like CN, while CD8-positive intraepithelial lymphocytes comparably appeared in both. Proton pump inhibitors (PPIs) were administered in five (71%) cases with MC-like features, and in three diarrhea improved after drug cessation. In IBD-like CN cases, eight (31%) received PPIs. Four patients received chemotherapy against malignancies. Four patients associated immune-related disorders. Microscopic appearance of CN also appeared in a remission state of ulcerative colitis (12/20 lesions).

Distal-type bronchiolar adenoma of the lung expressing p16INK4a - morphologic, immunohistochemical, ultrastructural and genomic analysis - report of a case and review of the literature.

Bronchiolar adenoma (BA) of the lung is a rare benign neoplasm. Because of a chest abnormal shadow indicated by health checkup, a 77-year-old female nonsmoker underwent computed tomography, revealing an 8 mm ground glass nodule in the peripheral field of the right lower lobe. Wedge resection of the nodule was performed, with a frozen diagnosis of primary lung adenocarcinoma. The localized, 8 × 4 × 3 mm-sized, jelly-like mass microscopically revealed a lepidic-growing lesion composed of ciliated columnar cells, mucous cells and basal cells surrounded by mucin pool. Neither nuclear atypia nor mitotic activity was noted. Immunohistochemically, the ciliated, mucous and basal cells were positive for TTF-1 and p16INK4a . Mucous cells were positive for napsin A and focally expressed MUC5AC. MUC6 was negative. Basal cells were positive for CK5/6, p40, p63 and podoplanin. Human papillomavirus genome was undetectable by in situ hybridization. Ultrastructurally, the bronchiolar epithelial tubules consisted of two layers, the inner nonciliated microvillous cells and the outer basal-like cells, and some of the inner cells were filled with mucin granules in cytoplasm. Molecular analysis of the tumor failed to show driver mutations. The final diagnosis was distal-type BA. The postoperative course was uneventful for 6 months.

Chronic Chagastic cardiomyopathy associated with membranoproliferative glomerulonephritis: Report of an autopsy case.

An autopsy case of chronic Chagas disease, a debilitating disorder caused by persistent infection by protozoa, Trypanosoma cruzi (Tr. cruzi), is reported. The patient was a 73-year-old Brazilian woman of Japanese descent, who had emigrated to Japan at the age of about 40 years. She died of chronic cardiac insufficiency about 8 years after the onset of cardiac symptoms. At autopsy, the heart showed typical features of chronic Chagastic cardiomyopathy: chronic lymphocytic myocarditis with extensive fibrosis and the formation of an apical aneurysm. The pathogenic protozoa were not detected in the cardiac tissue. The kidney showed typical features of membranoproliferative glomerulonephritis (MPGN). On the basis of experimental data which suggested that chronic infection of Tr. cruzi could elicit immune complex-mediated glomerulonephritis, we considered that the chronic persistent infection by Tr. cruzi contributed to the pathogenesis of MPGN in this patient.

Nuclear staining of claudin-18 is a new immunohistochemical marker for diagnosing intramucosal well-differentiated gastric adenocarcinoma.

Diagnosis of gastric adenocarcinoma using small biopsy samples is occasionally difficult. Various markers have been employed for improving the diagnostic accuracy, but there remains room for improvement. A total of 129 endoscopically biopsied samples were studied, consisting of 104 intramucosal tubular adenocarcinomas, 24 non-cancerous lesions and one cancer sample originally suspected of non-cancer but revised as cancer after immunostaining. We evaluated the association between histopathology and immunohistochemical expression of MUC1, HER2, p53, CEA, E-cadherin, ß-catenin and claudin-18. Regarding ß-catenin and claudin-18, not only membranous expression (ß-catenin(M) and claudin-18(M)) but also nuclear expression (ß-catenin(N) and claudin-18(N)) were analyzed. When subtyped with mucin core protein expression, the gastric-type cancers dominantly expressed claudin-18(M), while claudin-18(N) was significantly encountered in intestinal- and mixed-types. Expression of MUC1 (P = 0.0010), HER2 (P = 0.0173), p53 (P = 0.0002), CEA (P = 0.0019) and claudin-18(N) (P < 0.0001) revealed significant correlation with gastric cancers. Negative correlation of claudin-18(M) (P = 0.0125) was also noted. MUC1 and p53 were negative in non-cancer lesions. The non-cancer group exceptionally expressed HER2 and ß-catenin(N). Membranous expression of E-cadherin was consistent in both groups. Logistic regression analysis showed that MUC1 (P = 0.0086), p53 (P = 0.0031), claudin-18(M) (P = 0.0158) and claudin-18(N) (P = 0.0190) were independently associated with gastric cancers. Nuclear expression of claudin-18 should be the novel diagnostic marker for gastric cancer.

Acute actinomycotic brain abscess in a patient with rheumatoid arthritis.

An autopsy case of acute actinomycotic brain abscess involving a patient with rheumatoid arthritis (RA) is reported. The patient was a 72-year-old man with a seven-year history of RA and pulmonary complications, who acutely developed dysarthria and dysphagia three days before death. Autopsy revealed a fresh, non-encapsulated abscess in the "late cerebritis" stage, measuring 2 cm in diameter, in the white matter of the right parietal lobe. A small number of tiny "sulfur granules" consisting of numerous filamentous bacilli were found within the abscess. The abscess had ruptured to the lateral ventricle and elicited ventriculitis, and mild acute purulent leptomeningitis was also observed. The lung showed chronic interstitial pneumonia/pulmonary fibrosis with bronchiectasis and emphysema, and large sulfur granules were found in the lumens of a few bronchi. Less than 5% of patients with actinomycotic infection develop central nervous system lesions, and actinomycotic brain abscesses make up only 0.6% of all brain abscesses. Actinomycotic brain abscesses usually pursue a protracted clinical course, and well-formed pyogenic membranes are commonly observed. The present case is exceptional in that the very early stage of the cerebral abscess formation was pathologically captured.

Fatal community-acquired Bacillus cereus pneumonia in an immunocompetent adult man: a case report.

BACKGROUND: Bacillus cereus is a gram-positive rod bacterium that is responsible for food poisoning. It is naturally widely distributed, and thus often contaminates cultures. Although it is rarely considered responsible, it can cause serious infections under certain conditions. However, lethal infections, especially in immunocompetent patients, are rare. CASE PRESENTATION: A healthy 60-year-old man developed community-acquired B. cereus pneumonia and alveolar hemorrhage unveiled by abrupt chest pain and hemoptysis with no other advance symptoms. B. cereus induced silent alveolar destruction without any local or systemic inflammatory response. Although the lesion resembled lung anthrax, there was no evidence of Bacillus anthracis toxin. CONCLUSIONS: Some isolates of B. cereus can cause anthrax-like fulminant necrotizing pneumonia in immunocompetent patients. If this type of B. cereus were used as a means of bioterrorism, it may be quite difficult to recognize as bioterrorism. We should keep B. cereus in mind as a potential pathogen of fulminant human infectious disease.

Transduced caudal-type homeobox (CDX) 2/CDX1 can induce growth inhibition on CDX-deficient gastric cancer by rapid intestinal differentiation.

Intestinal metaplasia induced by ectopic expression of caudal-type homeobox (CDX)2 and/or CDX1 (CDX) is frequently observed around gastric cancer (GC). Abnormal expression of CDX is also observed in GC and suggests that inappropriate gastrointestinal differentiation plays essential roles in gastric tumorigenesis, but their roles on tumorigenesis remain unelucidated. Publicly available databases show that GC patients with higher CDX expression have significantly better clinical outcomes. We introduced CDX2 and CDX1 genes separately into GC-originated MKN7 and TMK1 cells deficient in CDX. Marked suppression of cell growth and dramatic morphological change into spindle-shaped flat form were observed along with induction of intestinal marker genes. G0-G1 growth arrest was accompanied by changed expression of cell cycle-related genes but not with apoptosis or senescence. Microarray analyses additionally showed decreased expression of gastric marker genes and increased expression of stemness-associated genes. Hierarchical clustering of 111 GC tissues and 21 non-cancerous gastric tissues by selected 18 signature genes based on our transcriptome analyses clearly categorized the 132 tissues into non-cancer, "CDX signature"-positive GC, and "CDX signature"-negative GC. Gene set enrichment analysis indicated that "CDX signature"-positive GC has lower malignant features. Immunohistochemistry of 89 GC specimens showed that 50.6% were CDX2-deficient, 66.3% were CDX1-deficient, and 44.9% were concomitant CDX2/CDX1-deficient, suggesting that potentially targetable GC cases by induced intestinal differentiation are quite common. In conclusion, exogenous expression of CDX2/CDX1 can lead to efficient growth inhibition of CDX-deficient GC cells. It is based on rapidly induced intestinal differentiation, which may be a future therapeutic strategy.

Expression of Gastric Markers Is Associated with Malignant Potential of Nonampullary Duodenal Adenocarcinoma.

BACKGROUND: Sporadic nonampullary duodenal epithelial tumors (NADETs) are uncommon, and thus their clinicopathological features have not been fully assessed. AIMS: In this study, we have analyzed a series of early sporadic NADETs, focusing on various immunohistological features. METHODS: We conducted a multicenter retrospective analysis of 68 patients with endoscopically resected sporadic NADETs. Associations between immunohistological features and clinicopathological features were statistically analyzed. RESULTS: The 68 patients consisted of 46 men (68%) and 22 women (32%) with a mean age of 60.7 ± 12.2 years (range 37-85 years). The 68 tumors were composed of 39 adenomas (57%) and 29 early-stage adenocarcinomas (43%). Duodenal adenocarcinomas were larger in size than adenomas and had papillary architecture in their pathological diagnosis with statistical significance. Duodenal adenocarcinomas also demonstrated a significantly higher expression of gastric markers (MUC5AC and MUC6) and a higher MIB-1 index. Duodenal adenomas were contrastively apt to express intestinal markers (MUC2, CDX1 and CDX2). Of the 68 cases analyzed, there were only 3 tumors positive for p53 staining, all of which were adenocarcinoma. When 7 submucosal invasive cancers and 21 intramucosal cancers were compared, submucosal invasion was positively associated with expression of MUC5AC. Also, submucosal invasion showed strong association with double-positivity of MUC5AC and MUC6. CONCLUSIONS: Our results indicate that immunohistochemical evaluation is useful for predicting malignant potential of NADETs, especially focusing on the expression of gastrointestinal markers.

Fulminant group A streptococcal infection without gangrene in the extremities: Analysis of five autopsy cases.

Five autopsy cases of fulminant group A streptococcal infection without gangrene in the extremities are presented. Clinical course of the fulminant illness was short (2-4 days). One pathological autopsy case was aged (86-years-old), and hemorrhagic cystitis was observed. The other four forensic autopsy cases were young (24-38 years-old) with the mean age of 32, and the primary infective lesions were located in the postpartum endometrium, tonsil and bronchus (2 cases). Systemic coccal dissemination with poor neutrophilic reaction was seen in two of five cases. Bilateral renal cortical necrosis was noted in three cases (including two with bacterial embolism). Hemophagocytosis, probably resulting from hypercytokinemia, was characteristic in three cases without bacterial embolism. Gram-positive cocci colonizing the hemorrhagic and necrotizing lesions were consistently immunoreactive for streptococcal antigens and Strep A (a carbohydrate antigen on group A streptococci). Neutrophilic reaction was mild in the primary infected foci. Clinicians should note that fulminant streptococcal infection (streptococcal toxic shock syndrome) in young and immunocompetent patients may not be associated with gangrene in the extremities. Autopsy prosecutors (diagnostic and forensic pathologists) must recognize the difficulty in making an appropriate autopsy diagnosis, particularly when bacterial embolism is not associated.

Nectin1 expression is frequently decreased in gastric cancers.

Gastric cancer (GC) is rich in many different histological types, but how the histological pattern is defined remains to be proved. The relation between GC histological types and the expression of nectin1, which is one of the cell adhesion molecules that composes adherens junction, has not been reported. According to a publicly available database of 406 GC patients, the median overall survival of Nectin1 high expression patients was 55.4 months and that of low expression patients was 25.6 months (P = 0.0246). Using surgically or endoscopically resected GC samples, nectin1 expression was analyzed by immunohistochemistry. Nectin1 expressed at adherens junction in all the normal epithelial cells. However, nectin1 expressed not at adherens junction but at apical membrane in epithelial cells in intestinal metaplasia. The expression pattern of nectin1 in intestinal type GC resembled to intestinal metaplasia. In order to analyze the difference in nectin1 expression between GC histological types, a total of 116 intestinal type GC and 33 diffuse type GC. The expression of necitin1 in diffuse type GC (3.0%) was remarkably decreased compared to that in intestinal type GC (65.5%) (P < 0.0001). In conclusion, this is the first report showing an association between nectin1 expression and histological subtypes of GC.

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.

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.