Profiling Microbial Communities in Idiopathic Granulomatous Mastitis.

Idiopathic granulomatous mastitis (IGM) is a rare and benign inflammatory breast disease with ambiguous aetiology. Contrastingly, lactational mastitis (LM) is commonly diagnosed in breastfeeding women. To investigate IGM aetiology, we profiled the microbial flora of pus and skin in patients with IGM and LM. A total of 26 patients with IGM and 6 patients with LM were included in the study. The 16S rRNA sequencing libraries were constructed from 16S rRNA gene amplified from total DNA extracted from pus and skin swabs in patients with IGM and LM controls. Constructed libraries were multiplexed and paired-end sequenced on HiSeq4000. Metagenomic analysis was conducted using modified microbiome abundance analysis suite customised R-resource for paired pus and skin samples. Microbiome multivariable association analyses were performed using linear models. A total of 21 IGM and 3 LM paired pus and skin samples underwent metagenomic analysis. Bray−Curtis ecological dissimilarity distance showed dissimilarity across four sample types (IGM pus, IGM skin, LM pus, and LM skin; PERMANOVA, p < 0.001). No characteristic dominant genus was observed across the IGM samples. The IGM pus samples were more diverse than corresponding IGM skin samples (Shannon and Simpson index; Wilcoxon paired signed-rank tests, p = 0.022 and p = 0.07). Corynebacterium kroppenstedtii, reportedly associated with IGM in the literature, was higher in IGM pus samples than paired skin samples (Wilcoxon, p = 0.022). Three other species and nineteen genera were statistically significant in paired IGM pus−skin comparison after antibiotic treatment adjustment and multiple comparisons correction. Microbial profiles are unique between patients with IGM and LM. Inter-patient variability and polymicrobial IGM pus samples cannot implicate specific genus or species as an infectious cause for IGM.

Further Characterization of Clinicopathologic Features of Cystic Neutrophilic Granulomatous Mastitis.

OBJECTIVES: Clinical and demographic features of cystic neutrophilic granulomatous mastitis (CNGM) have not been fully explored due to the rarity of the disease. Herein we studied clinicopathologic characteristics of CNGM in a sizable hospital-based cohort. METHODS: A case-control study was performed to compare clinicopathologic characteristics between patients with CNGM and granulomatous mastitis other than CNGM and between CNGM with and without Corynebacterium identification. RESULTS: Cases of CNGM (n = 31) and non-CNGM (n = 30) were included. Compared with the non-CNGM group, patients with CNGM were statistically significantly younger (median age: 38 vs 43 years), were less likely to be smokers (9% vs 40%), were more likely to have a painful lesion (97% vs 77%) or a larger mass-like lesion (median size: 4.6 vs 1.9 cm), and tended to have a higher Breast Imaging Reporting and Data System score in radiologic studies (score ≥4: 81% vs 53%), positive Corynebacterium identification results (36% vs 0%), and a longer resolving time (12 vs 6 months; all P values for above comparisons <.05). Among CNGM cases, patients with and without Corynebacterium identification shared a similar clinicopathologic profile. CONCLUSIONS: Our study further demonstrated that CNGM is a unique infectious disease with distinct clinicopathologic features.

Detection of Corynebacterium kroppenstedtii in Granulomatous Lobular Mastitis Using Real-Time Polymerase Chain Reaction and Sanger Sequencing on Formalin-Fixed, Paraffin-Embedded Tissues.

CONTEXT.­: Associations between granulomatous lobular mastitis (GLM) and Corynebacterium kroppenstedtii have been reported since 2002, but large-scale studies to assess the actual prevalence of this bacterium in GLM have not been performed. OBJECTIVE.­: To assess the prevalence of C kroppenstedtii in GLM using real-time polymerase chain reaction and Sanger sequencing. DESIGN.­: We analyzed formalin-fixed, paraffin-embedded tissues from 67 cases of GLM by sequential DNA amplification and sequencing to assess the rate of C kroppenstedtii detection in GLM. A retrospective analysis including patient demographics, history of pregnancy and lactation, clinical signs and symptoms, radiographic findings, histologic pattern, Gram stain results, and microbial cultures was performed on 67 cases of GLM. In addition, 10 cases of nongranulomatous breast abscess were included as controls. RESULTS.­: C kroppenstedtii 16S rRNA SYBR real-time polymerase chain reaction was positive on formalin-fixed, paraffin-embedded tissues from 46 of 67 (68.7%) GLM cases, while all control cases were negative. Among the positive cases, the majority showed features of cystic neutrophilic granulomatous mastitis. CONCLUSIONS.­: C kroppenstedtii was highly prevalent in GLM cases and was not found to be associated with nongranulomatous breast abscess in our study (P < .001).

Granulomatous mastitis caused by Rickettsia species.

Granulomatous mastitis is a rare inflammatory disease of varying etiology. Tuberculosis and cystic neutrophilic granulomatous mastitis caused by Corynebacterium are the best-established infectious examples. Despite the increasing incidence of Rickettsia-related diseases worldwide, granulomatous inflammation of breast parenchyma caused by Rickettsia has not yet been reported. We present a unique case of bilateral granulomatous mastitis documented with mammography, magnetic resonance imaging and core-needle biopsy. The rickettsial etiology of the disease was proved with specific immunohistochemistry and confirmed with DNA extraction, PCR and serology. The lesions completely resolved after a full-course tetracycline treatment. This case report widens the knowledge about the possible clinical manifestations of Rickettsia infection and adds a new bacterium to the list of etiological factors causing granulomatous mastitis.

Cystic neutrophilic granulomatous mastitis - a review of 12 consecutive cases.

AIMS: Cystic neutrophilic granulomatous mastitis (CNGM) is an uncommon but increasingly recognised cause of mastitis, often associated with Corynebacterium ssp. infection. We studied the histopathological and clinical features of CNGM in a Canadian setting, and the work-up required to identify pathogenic microorganisms. METHODS AND RESULTS: A retrospective search for breast specimens with abscess, acute, chronic and/or granulomatous inflammation from 1998 to 2018 was performed. Haematoxylin and eosin slides were reviewed for typical histological features of CNGM. Histochemically stained slides for microorganisms were also reviewed. Repeat Gram stains were performed if initially negative. Electronic medical records were abstracted for microbiology results and relevant clinical data. Twelve cases were identified. All were female, aged 25-57 years, mainly Caucasian, with one Venezuelan and two of Chinese ethnicity. Most were parous (10 of 12); five of 12 had an endocrinopathy. Bacteria were identified in one or more specimens from eight of 12 patients; additional Gram stains revealed organisms in four of 12 cases. Of four bacterial cultures, one grew Corynebacterium kroppenstedtii. 16S polymerase chain reaction for three samples was negative. Two patients had multiple breast biopsies, showing early palisaded granulomas followed by classic features of CNGM. The patients had various management approaches, including surgery and antimicrobials. CONCLUSIONS: CNGM may present as palisaded granulomatous inflammation, without the expected 'cystic' pattern, suggesting that there is an evolution of histomorphology with this infection. Most patients with CNGM are parous, and there may be an association with endocrinopathies. Application of multiple Gram stains increases the yield of microorganism identification. Recognition of CNGM in breast biopsies and collaborative communications are essential to direct appropriate therapy.

Cystic neutrophilic granulomatous mastitis: an update.

Cystic neutrophilic granulomatous mastitis (CNGM) is a rare subtype of granulomatous mastitis with a highly distinct histological pattern often associated with Corynebacterium species. CNGM is characterised by suppurative lipogranulomas that are composed of central lipid vacuoles rimmed by neutrophils and an outer cuff of epithelioid histiocytes. Some of the lipid vacuoles may contain sparse, rod-shaped, gram-positive bacilli that can be easily missed or dismissed. The surrounding mixed inflammatory infiltrate contains Langhans-type giant cells, lymphocytes and neutrophils. CNGM occurs in reproductive age women with a history of pregnancy and typically presents as a palpable mass that can be painful. CNGM has many mimickers, most significantly breast carcinoma. In many cases, CNGM has significant pathological and clinical overlap with other forms of granulomatous mastitis. Given the association with Corynebacterium species, early diagnosis of CNGM is essential in offering patients the most appropriate treatment. Prolonged antibiotic therapy specifically directed to corynebacteria is required, sometimes even beyond resolution of clinical symptoms. This comprehensive review of the existing literature on CNGM describes clinical-pathological features, microbiological findings, challenges associated with the microscopic differential diagnosis, clinical implications of this diagnosis and emerging treatment options. Morphological criteria and suggested comments to convey the degree of diagnostic certainty are also proposed for standard pathology reporting.

"Thick Section" Gram Stain Yields Improved Detection of Organisms in Tissue Sections of Cystic Neutrophilic Granulomatous Mastitis.

OBJECTIVES: Cystic neutrophilic granulomatous mastitis (CNGM) is an uncommon subtype of granulomatous mastitis featuring epithelioid histiocytes/neutrophils rimming discrete cystic spaces. When present, gram-positive bacteria (typically Corynebacterium species) are identified within these cystic spaces, although they can be difficult to appreciate on tissue sections. METHODS: Based on pilot gram-stained tissue sections of CNGM cases cut on 6 µm thickness (in which bacterial organisms were more readily identifiable) instead of the traditional 4 µm, a formal comparative analysis of 19 CNGM cases was performed on parallel 6-µm ("thick") vs 4-µm sections from one representative block per case. RESULTS: Biopsies (n = 17) and excisional specimens (n = 2) from 19 CNGM cases were included. Gram-positive palisaded rods were identified in seven cases using 4-µm sections and in 11 cases using 6-µm sections (sensitivity of 37% vs 58% in identifying organisms). Among all seven cases of organisms seen on the 4-µm section Gram stain, the paired 6-µm section Gram stain showed a higher number of and more readily identifiable bacteria. CONCLUSIONS: Thick section Gram stain of representative tissue blocks performed at 6 µm improves both detection rate and ease of identification of gram-positive organisms in CNGM.

Cystic Neutrophilic Granulomatous Mastitis: A Clinicopathological Study With 16s rRNA Sequencing for the Detection of Corynebacteria in Formalin-Fixed Paraffin-Embedded Tissue.

Cystic neutrophilic granulomatous mastitis (CNGM) is a histologically characterized variant of granulomatous lobular mastitis that is associated with lipophilic Corynebacterium species. It remains a largely underrecognized entity in India. Our aim was to study CNGM in the Asian Indian population and explore if 16s rRNA sequencing could be used on formalin-fixed paraffin-embedded (FFPE) tissue to identify the causative organism. We studied 24 cases with histological features of CNGM with hematoxylin and eosin, Gram, Ziehl-Neelsen, and Periodic acid-Schiff stains. Tuberculosis-polymerase chain reaction and 16s rRNA gene sequencing on DNA extracted from FFPE was attempted (N = 23). Gram-positive bacilli were seen in 20/24 cases. Routine culture with prolonged incubation yielded Corynebacterium species in 8 cases; 7 of these cases were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species identification. C matruchotti was identified in one case by BD Phoenix. MALDI-TOF MS identified the remaining 7 cases as C kroppenstedtii (N = 4) and C tuberculostearicum (N = 2), with no identification in one. Corynebacteria were identified by 16s rRNA sequencing on DNA extracted from FFPE in 12/23 cases using a primer targeting the V5-V6 region that was found to be more conserved in Corynebacterium species. All cases were negative for the diagnosis of tuberculosis. CNGM can be identified by routine stains. Culture using routine media with prolonged incubation is often adequate to isolate the organism. 16s rRNA sequencing on DNA extracted from FFPE tissue can help make an etiological diagnosis in some cases where only paraffin blocks are available.

Granulomatous Mastitis and Factors Associated with Recurrence: An 11-Year Single-Centre Study of 113 Patients in Singapore.

BACKGROUND: Granulomatous mastitis (GM) is an inflammatory breast disease of unknown aetiology. It poses diagnostic and therapeutic challenges with myriad forms of clinical presentation, varying results to treatments and propensity to recur. This study aims to look at clinical and treatment factors that predispose to recurrence of GM. METHODS: We performed a retrospective review of 113 patients in our unit with histologically proven GM from 2006 to 2016. Demographic, clinical, treatment and outcomes data were collected and analysed. RESULTS: Eighty-nine patients were treated with antibiotics (78.8%), 79 (69.9%) with steroids and 23 (20.4%) patients underwent surgery. Twenty (17.7%) patients had recurrence. Patients who presented with inflammatory signs and symptoms had increased odds of having subsequent recurrence: skin changes (1.50), pain (2.00), fistula (4.39) and antibiotic treatment (6.65). Four patients (20%) with recurrence had positive bacterial cultures. All 4 grew Corynebacterium. Patients with Corynebacterium infection had a 2.64 times higher risk of recurrence. Surgery did not preclude recurrence. There was a 70% (7/10) penicillin resistance rate in our patients with positive cultures for Corynebacterium. CONCLUSION: Initial presentation with inflammatory signs and symptoms may confer increased risk of recurrence, warranting closer monitoring. Corynebacterium infection may play a part as a causative factor and risk factor for recurrence. Non-penicillin antibiotics should be considered as first-line antibiotics for patients presenting with inflammatory changes. Further prospective studies with larger patient populations might reveal information on the aetiology of GM and result in the development of a more standardized and effective treatment regimen.

Corynebacterium kroppenstedtii: a challenging culprit in breast abscesses and granulomatous mastitis.

PURPOSE OF REVIEW: Corynebacterium kroppenstedtii is a difficult pathogen associated with granulomatous mastitis and recurrent breast abscesses. Despite over a dozen studies, there is no guidance on surgical interventions, steroid use, or dosing or duration of antibiotic treatment. RECENT FINDINGS: Initially seen in predominantly Maori and Pacific Islander multiparous, postlactating women, C. kroppenstedtii breast infection has since been reported throughout the world, including in nulliparous women. Additionally, emerging data suggest that hyperprolactinemia is a modifiable risk factor for these infections. This article reviews a patient case and data from 87 other cases to compile current best practices for diagnosis, treatment, and monitoring, and provides areas for future study. SUMMARY: In cases of granulomatous mastitis and breast abscess, especially if recurrent, infection with C. kroppenstedtii should be considered. Microbiologists should be alerted to the specialized growth conditions and tools needed for appropriate culturing, identification, and antibiotic susceptibility testing. Clinicians should utilize a multimodal approach with surgical and antibiotic treatment to maximize clinical cure and reduce recurrence.

[Granulomatous lobular mastitis: a clinicopathological analysis of 300 cases].

Objective: To investigate the clinicopathologic features and possible causes of granulomatous lobular mastitis(GLM). Methods: Three hundred cases of GLM were collected from surgical specimens diagnosed at Longhua Hospital, Shanghai University of Traditional Chinese Medicine from January 2015 to November 2017. Morphologic features were reviewed using HE staining. A total of 116 cases were investigated by Gram staining. The expression of CD3, CD20, CD68, IgG, IgG4, CD38 and CD138 was detected by immunohistochemical staining. Results: The age of the patients was 23 to 47 years and the median age was 32 years. All patients were female, 96.7% (290/300) had a history of lactation.There were 143 cases of left breasts, 138 cases of right breast and 19 cases of bilateral breasts. Serum prolactin increased in 39.7%(119/300) patients. Within 15.7%(47/300) of patients were associated with nodular erythema or joint swelling and pain of the lower extremities. Pathological observation showed that lobular-centric suppurative granulomatous inflammation, accompanied by dilatation of intralobular and interlobular ducts. There were 16 cases accompanied with duct ectasia. Immunohistochemistry showed CD3-positive lymphocytes were more than CD20-positive lymphocytes in the peripheral aggregation zone of neutrophils within granulomatous lesions. Gram positive bacteria were found in the lipid vacuoles of the 51.7%(60/116) patients. Conclusions: GLM has distinctive histologic features. It may be related to corynebacterium infection, or accompanied by the increase of serum prolactin and erythrocyte sedimentation rate. The age, location and history of the disease are importance in the diagnosis and differential diagnosis.

Pathogens in patients with granulomatous lobular mastitis.

OBJECTIVE: Granulomatous lobular mastitis (GLM) is a rare inflammatory disease of the breast that clinically mimics breast cancer. However, its etiology is not completely defined. The purpose of this study was to systematically study the bacteriology of GLM using advanced detection technology. METHODS: Paraffin-embedded tissue from patients with GLM was collected. DNA was extracted from the samples and analyzed using next-generation sequencing (NGS) technology, and the data were processed using bioinformatics analyses. RESULTS: A total of 40 patients were recruited into the study. A bioinformatics analysis revealed that a total of 17 genera or 19 species of pathogens were present in 39 of the GLM patients (97.5%). These included bacteria, fungi, and Mycobacterium tuberculosis complex group. Bacteria were found in 39 of the patient cases, while fungi were present in five. Only one case tested positive for M. tuberculosis complex. In addition, a single genus of pathogen was found in nine patients (23.1%), whereas 30 patients (76.9%) tested positive for multiple pathogens. CONCLUSIONS: This study profiled the microbiota of patients with GLM using NGS technology, which provides more useful information for establishing patient treatment plans.

Coryneform Bacteria in Granulomatous Lobular Mastitis: Morphological Diagnosis in Breast Biopsies.

Granulomatous lobular mastitis is a rare disease whose origin is still unknown and shows an increase in its frequency. Morphological, microbiological, and molecular biology studies have linked this disease to lipophilic and fastidious corynebacteria, suggesting its possible infectious etiology. This series describes and reviews in detail the distinctive morphological characteristics of the bacteria present in the granulomas of this disease, the usefulness of histochemical techniques for their identification, and our proposal for a tissue quantification score for the bacteria. The MacCallum-Goodpasture method of Gram's stain turned out to be the gold standard for examination, but we also highlight the efficiency of hematoxylin and eosin stain when it is exhaustively examined as well as the Grocott stain to evaluate the bacterial pleomorphism method, which is often underutilized.

Cystic neutrophilic granulomatous mastitis: The Cleveland Clinic experience with diagnosis and management.

Granulomatous mastitis is an uncommon inflammatory disease that typically presents with painful breast lesions. Recent publications have brought to light a specific subset of granulomatous mastitis patients with a distinct histological pattern of disease termed, "cystic neutrophilic granulomatous mastitis" (CNGM). Although many cases of granulomatous lobular mastitis have been thought to be idiopathic, this rare subset of an uncommon disease has been linked to infections with Corynebacterium species. Herein, a cohort of CNGM patients from a large, tertiary care, North-American, academic medical center is presented. Correlative demographic, clinical, radiographic, pathologic, microbiologic, management, and outcomes data are provided. Collaborative communication between specialists to accurately diagnose and manage these patients is essential to decreasing potential morbidity.

Idiopathic granulomatous mastitis: a 10-year study from a multicentre clinical database.

Idiopathic granulomatous mastitis (IGM) is an uncommon, chronic inflammatory breast disease with elusive aetiology, simulating malignancy clinically and radiologically. Here we present our 10-year review on a region-wide multicentre IGM database. A retrospective study was performed on a prospectively maintained database from three University affiliated hospitals in Hong Kong and Shenzhen, China. All patients with biopsy proven IGM were included while patients with positive culture of Mycobacterium tuberculosis were excluded. Disease recurrence rate and its prognosticators were evaluated. A total of 102 patients were included between January 2007 and December 2017. Median age was 33 years (range 20-54). Most patients presented with painful inflammatory mass (n = 57); median size at presentation was 37 mm (6-92 mm). Sixty-three patients had bacterial culture performed on the pus sample: eight patients had Corynebacterium kroppenstedtii while four had Corynebacterium species not otherwise specified. Seventy-seven (75.5%) patients received conservative treatment with oral corticosteroid (±antibiotics) and drainage only, while 25 (24.5%) patients received breast lump excision after initial medical treatment. Twelve (11.8%) patients developed recurrence after a median follow-up interval of 14 months (4-51 months). Univariate analysis revealed that abscess on presentation, history of smoking, and presence of C. kroppenstedtii were significant prognosticators for recurrence. Subsequent multivariate analysis with logistic regression revealed cigarette smoking and isolation of C. kroppenstedtii as independent risk factors for disease recurrence (p < 0.05). In conclusion, IGM is uncommon with a recurrence rate of 12%, especially in patients with history of smoking and isolation of C. kroppenstedtii.

Cytomorphological features of cystic neutrophilic granulomatous mastitis on ThinPrep.

Granulomatous mastitis (GM) is an uncommon entity and challenging to distinguish from breast carcinoma, both clinically and upon imaging. Cystic neutrophilic granulomatous mastitis (CNGM) is a histologically distinct GM with clear "cystic" spaces (CS) surrounded by neutrophils and may contain Gram-positive bacilli (such as Corynebacterium species). Cytologic features of GM, in particular CNGM, on ThinPrep (TP), are limited. We reviewed the cytological features of GM with a special focus on CNGM on TP. Ten cases with a concurrent/recent FNA and histologic diagnosis of GM or CNGM, or positive culture for Corynebacterium kroppenstedtii, were identified over an 11-y period (2007-2017). A double-blinded review of the TP slides was performed by two independent cytopathologists. Data were analysed using Fisher's exact test. Six cases were histologically proven CNGM and 1 FNA only case had a positive culture for C. kroppenstedtii. Three had a histological diagnosis of GM. TP from the 7 CNGM cases demonstrated CS, including 1 case with a cellblock (CB) study showing well-formed clear CS, while none of the non-CNGM cases had CS (P = .01). Bacteria were identified within CS in 5 of 7 cases of CNGM. Multinucleated giant cells, granulomas, histiocytes, and neutrophils were variably observed. All histologically confirmed cases of CNGM showed distinct CS on TP. Cellblock study is a valuable adjunct to evaluate CNGM and for ancillary studies. Although our study was limited by a small sample size, our findings necessitate the need for studies with a larger cohort.

Cystic neutrophilic granulomatous mastitis: A clinicopathologic study of a distinct entity with supporting evidence of a role for Corynebacterium-targeted therapy.

Cystic neutrophilic granulomatous mastitis (CNGM) is a distinct histopathologic entity characterized by neutrophilic and granulomatous inflammation surrounding clear cystic spaces. Rare gram-positive bacilli are sometimes identified within these cystic spaces. Studies in the literature have identified these gram-positive bacilli to be Corynebacterium species. We describe the clinicopathologic features of 7 cases of CNGM, including a case with evidence of Corynebacterium amycolatum. Patients were young to middle aged parous women ranging in age from 28 to 53 years (median age: 41 years). Gram-positive bacilli were identified in 4 cases, all within cystic spaces. Microbial culture from a 41-year old Hispanic woman grew Corynebacterium species on multiple occasions and Corynebacterium amycolatum was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on two separate occasions. Antibiotic susceptibility testing performed both times showed resistance to multiple antibiotics and susceptibility to vancomycin. Follow-up of all patients (range 3-12 months, median 6 months) showed a widely variable clinical course and varying response to a variety of treatment modalities. Five of the seven CNGM patients were parous, reproductive-aged Hispanic women who were born outside of the United States. Our findings further support the association of CNGM with corynebacteria and gram-positive bacilli. Furthermore, this study shows that Corynebacterium amycolatum, a nonlipophilic and multidrug-resistant corynebacterium can be associated with CNGM, hence the need for targeted antibiotic therapy. We propose identifying corynebacteria to the species level and performing antibiotic susceptibility testing in patients with CNGM because of the varied susceptibility testing profile that has been reported among different species of corynebacteria.

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.