Methanamine Silver Stain Research Articles

Histochemical Demonstration of Fungi in Breast Diseases at University of Maiduguri Teaching Hospital-North Eastern Nigeria

Breast disease is a global public health concern, and recent research suggests that fungal infections may be linked to its development and progression. This study aims to investigate the presence of fungi in formalin-fixed paraffinembedded (FFPE) breast tissue samples diagnosed with various breast diseases, including breast cancer, using histological staining techniques. The study design was a retrospective cross-sectional study that used convenience sampling to retrieve 49 breast tissue blocks diagnosed with various breast diseases from the Histopathology archive of the University of Maiduguri Teaching Hospital, Nigeria. The samples were re-embedded with paraffin wax, sectioned, and stained using Haematoxylin and Eosin and Grocott Methanamine Silver Stain. The data collected was analyzed descriptively and using chi-square tests. Fibroadenoma was the most common diagnosis in the study (69.3%), followed by Chronic Granulomatous Mastitis (18.4%) and breast cancer (12.2%). The highest frequency of Fibroadenoma diagnosis was in the age range of 30-39, whereas the highest frequency of breast cancer diagnosis was in the age range of 20-29. The study found that fungal infections were present in breast tissue samples, and the highest percentage of positive Gomori Methenamine Silver (GMS) stain test results were found in breast disease cases with breast cancer with a significant p value of 0.0175. This study provides evidence of a potential association between fungal infections and breast diseases in North Eastern Nigeria. Further research is needed to fully understand the mechanisms underlying this association and its implications for breast cancer prevention and treatment.

Evaluation of Histopathological Effects of Smoked Marijuana on Albino Rats and Its Oxidative Stress Indices

Smoking marijuana (cannabis Savita), a psychoactive substance may result in side effect on living cells of the system and oxidative stress may meditate some of these effects. This study aims to evaluate the acute and chronic histopathological effect on wistar albino rat as well as oxidative stress indices and some of the haematological parameters evaluation. This study was conducted at the animal house of Ebonyi State University, Presco campus Abakaliki. The experimental animals were share into control group, acute and chronic administered group which was housed in the temperature and humidity controlled facility of 22°C+1, standard laboratory mice chow diet with tap water freely available at ad libitum to the animals. The weight of the male wistar albino rat were taken twice daily which range from 150g to 200g after which was exposed to smoke of burnt wrapped 2mg of marijuana morning and evening for 21 days for experimental animals in group B and 42 days for group C animals respectively. The acute histopathological effect and that of chronic of the lungs of smoked marijuana wistar albino rat displayed histomorphological changes with Haematoxylin and Eosin, methanamine silver staining and phosphotungstic acid staining method when compared with the control lung sections that shows normal histomorphological pattern with the present of alveolar pneumocytes and alveolar space. Evaluation of the lungs of smoked marijuana rat shows increased MDA and decreased level of CAT, SOD, GSH and GPx but examining some of the haematological parameters show elevated Twbc and platelet while packed cell volume decreased the level as compared with control animals. I hereby concluded that marijuana smoked is a potent source of cellular oxidative stress that contributes significantly to cellular injury, dysfunction, and pulmonary edema as well as histomorphological changes from the lungs of marijuana smokers and disorder in some of the haematological parameters.

Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis.

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori’s methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.

Laryngeal histoplasmosis mimicking glottic cancer in an immunocompetent host

We report an interesting case of laryngeal histoplasmosis which clinically mimicked a malignant glottic growth and is unusual in that it involved an immunocompetent host. Also, an isolated laryngeal involvement is very rare. A 63 year old farmer presented with complaints of hoarseness of voice. Indirect layngoscopy revealed a friable growth in the middle of the right vocal cord. Histopathological examination of biopsy taken from the growth showed presence of numerous tiny intracellular organisms within collections of macrophages and epithelioid cells. Confirmation was done with periodic acid Schiff and Gomori’s methanamine silver stain. The patient was administered amphotericin B 1 mg/kg body weight for 2 weeks followed by itraconazole 200 mg, twice a day for 8 weeks and discharged on oral itraconazole. A dramatic improvement in voice quality was seen in follow-up after two months. Repeat endoscopy showed regression of growth. The otolaryngologist should be aware of the existence of laryngeal histoplasmosis and consider it as one of the differential diagnosis when a patient shows signs of granulomatous inflammation or laryngeal masses, to perform specific diagnostic tests for fungi and provide optimal and timely treatment, avoiding unnecessary aggressive interventions.

MON-021 IMMUNOHISTOLOGICAL SPECTRUM OF CRESCENTIC GLOMERLONEPHRITIS, A SINGLE CENTER STUDY IN BANGLADESH

Crescentic glomerulonephritis (GN) is defined as presence of crescents in >50% of glomeruli sampled for renal biopsy. This is a critical diagnosis with clinical implications that ideally is communicated to the referring physician immediately. There is limited data on the etiology, clinical and histopathological spectrum of crescentic glomerulonephritis (CrGN) in Bangladeshi population. All the crescentic glomerulonephritis diagnosed histologically at Armed Forces Institute of Pathology, Dhaka irrespective of age and sex are included in the study. The study duration was from January 2017 to June 2018. The renal biopsy samples were undergone H&E, PAS, Masson trichrome and Methanamine silver staining. For each case direct immunofluoresnece study was carried out for IgG, IgM, IgA, C3, C1q, Kappa and Lambda. Depending on clinical, biochemical, serological, histological and immunofluorescence evaluation crescentic glomerulonephritis was classified. During stipulated period 21 crescentic glomerulonephritis were diagnosed which comprises 2.56% out of total 819 non transplant renal core biopsy patients. Mean age was 27.238 +14.928 the largest age group (8/21) being paediatric age group (<16 yrs). The male female ratio was 0.75. The most common presentation was rapidly progressive glomerulonephritis (RPGN) (7/21), followed by generalized body swelling (6/21). On urine examination 19/21 patients had haematuria and 7/21 patients had massive proteinuria (>3.0 Gm/24 hrs). The mean serum creatinine level was 5.65 mg/dl (1.3 to 18.85 mg/dl). Serum C3 & C4 level was found low in 6/18 and 2/15 cases respectively. ANA was positive in 2/21 cases and among those 1 was anti dsDNA positive and labeled as systemic lupus erythematosus (SLE). Among 21 cases 01 could not be classified due to lack of glomerulus during direct immunofluorescence (DIF) study. Pauciimmune crescentic glomerulonephritis (PI-CGN) was 10 (50%), Immune complex mediated crescentic glomerulonephritis (IC-CGN) was 9 (45%) and anti glomerular basement membrane disease (anti GBM disease) was 01(5%). Among the PI-CGN, 5 (50%) were ANCA positive and 03 (30%) were ANCA negative, for other 02 PI-CGN cases ANCA report was not available. Among ANCA positive PI-CGN 01 had associated ANA positivity. Among IC-CGN, 02 were crescentic IgA, 01 was crescentic lupus nephritis, 01 was isolated C3 glomerulonephritis, 03 were membranoproliferative glomerulonephritis with diffuse crescent formation and rest 02 were IC-CGN of unknown reason. The glomerular crescents formation was 80.33% in PI-CGN while it was 61.82% among IC-CGN cases and 66.67% in anti GBM glomerulonephritis. Among IC-CGN, 4/9 (44.44%) cases needed dialysis on admission while 5/10 (50%) PI-CGN cases were managed during admission by dialysis. The only anti GBM disease was tried to be managed by frequent dialysis but failed and plasmapheresis was advised but could not be instituted who ultimately succumbed to death following severe haemoptysis. In our study commonest cause of crescentic glomerulonephritis was pauci-immune crescentic glomerulonephritis followed by immune complex mediated glomerulonephritis. While one case of anti GBM glomerulonephritis was found that was possibly a case of Good pasture syndrome.

Subcutaneous phaeohyphomycosis in an immunocompetent Individual: A case report

Phaeohyphomycosis is a rare mycotic infection caused by various heterogenous groups of phaeoid (dematiaceous) fungi involving the skin and subcutaneous tissue. Common clinical manifestations are subcutaneous abscesses or cystic swellings. Here, we report a case of subcutaneous phaeohyphomycosis presenting as multiple asymptomatic cystic swellings over the hands and feet without any predisposing factors. Histopathology showed granulomatous inflammation and special stain with Grocott's methanamine silver stain revealed broad pigmented hyphae. Culture showed black-colored colonies identified as Exophiala jeanselmi. The patient was treated with surgical excision of the lesions.

Pneumocystis pneumonia in HIV patients: a diagnostic challenge till date.

HIV has become a major health problem in India, patients commonly succumb to opportunistic infections (OIs), respiratory infections being an important cause of morbidity and their accurate diagnosis is still a challenge. Our aim was to study the occurrence of Pneumocystis pneumonia (PCP) in HIV/AIDS patients with respiratory complaints attending ART clinic and to compare various diagnostic methodologies. One hundred and twenty five HIV/AIDS patients presenting with respiratory symptoms like cough, fever, breathlessness etc, were enrolled, and induced sputum samples were collected. Samples were homogenized using glass beads and Dithiothretol. Smears were prepared and examined by Immunoflourescent staining (IFAT), Gomori methanamine silver staining (GMSS), Toludine blue O staining (TBO) and Giemsa staining for Pneumocystis jiroveci. Among the 125 patients who presented with respiratory complaints, 34 cases (27.2%) were diagnosed as having PCP. All 34 cases were detected by IFAT followed by GMSS, Giemsa and Toludine blue O staining in decreasing order. The mean CD4 count was 67.27cells/μl. PCP has become an important health problem in HIV/AIDS patients with low CD4 counts in India. IFAT remains the most sensitive method for the detection of this uncultivable organism. In resource poor settings where an immunoflourecent microscope is not available, diagnosis of PCP still remains problematic.

Pathology of mycotic tracheitis in poultry: A case report

A case of mycotic tracheitis in an adult Rhode Island Red bird of about 20 weeks of age is described here. Necropsy revealed multiple circumscribed granulomatous nodules in the lungs and caseous plugs in the trachea. Microscopically there were fungal hyphae penetrating the tracheal mucosa together with a caseative plug having central necrotic mass adhering to the tracheal wall, foci of severe congestion and hemorrhage, fungal granuloma surrounded by mononuclear cell infiltration, giant cell, fungal hyphae and fibrous tissues were recorded. The fungal hyphae were also demonstrated by Grocott's methanamine silver stain.

Primary Histoplasmosis of Larynx Mimicking as Laryngeal Carcinoma

ABSTRACT Granulomatous diseases caused by infectious agents are being encounted more frequently nowadays. Infection by Histoplasma capsulatum is one such granulomatous disease. Isolated laryngeal histoplasmosis is a rare clinical entity. Because of its simulation with malignancy, it needs to be included in the differential diagnosis of neoplasm, both benign and malignant. We report an extremely rare case of isolated histoplasmosis, presenting as a primary infection of the larynx with no other systemic involvement in a 57 years old immunocompetent male. He presented with progressive hoarseness of voice, difficulty in breathing and mild dysphagia. Indirect laryngoscopy revealed multiple exophytic nodular lesions in endolaryngeal structures. The patient then underwent direct laryngoscopy biopsy under general anesthesia and the histopathological examination of the biopsy revealed chronic granulomatous inflammation, caused by histoplasma which was confirmed by using special Gomori's methanamine silver stain for fungi. Though it appeared like cancer clinically; biopsy confirmed the final diagnosis. The histopathologist must be informed about the possibility of histoplasmosis, because special dyes have to be used to confirm this diagnosis. The treatment is mainly medical with amphotericinB which remains the gold standard for treatment of this disease, although other drugs have also been used. How to cite this article Bist SS, Sandhirr S, Shirazi N, AgrawalV, Bharti B. Primary Histoplasmosis of Larynx Mimicking as Laryngeal Carcinoma. Int J Phonosurg Laryngol 2015;5(1):28-31.

Fungal extracts detected in eosinophilic chronic rhinosinusitis induced cytokines from the nasal polyp cells

Objectives/HypothesisThe role of fungi in chronic rhinosinusitis (CRS) is still controversial. The present study was conducted to detect and identify fungal species from the nasal polyp tissues of eosinophilic and noneosinophilic CRS, and to determine the role of fungal antigens in cytokine production.Study DesignProspective study.MethodsThirty-five specimens of nasal polyps were collected from patients with CRS and examined for fungus using culture, histology, and polymerase chain reaction analysis. The secretion of 14 cytokines stimulated by fungal extracts using dispersed nasal polyp cells (DNPCs) was determined by multiplex immunoassay.ResultsThere was no microbiological growth (including fungus) in the cultures of homogenized nasal polyps. Furthermore, Grocott methanamine silver staining for all nasal polyps showed no fungal bodies. Sixteen of 35 samples of the nasal polyps showed amplification of fungal DNA. In none of the mucosa of the sphenoid sinus was fungal DNA detected. The number of eosinophils in the nasal polyps in which fungal DNA was detected was significantly higher than in the nasal polyps in which fungal DNA was not detected (P < .01). The extract of fungus enhanced the secretion of eosinophil-associated cytokines such as interleukine (IL)-5, IL-13, IL-17A, and RANTES (regulated on activation normal T-cell expressed and secreted), and proinflammatory cytokines such as IL-6, IL-8, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor from DNPCs.ConclusionsThe present study offers direct evidence supporting that fungal elements modify the inflammatory response in the nasal polyps of eosinophilic CRS.Level of Evidence: NA

Clinicopathologic assessment of Candida colonization of oral leukoplakia.

Leukoplakia is the most common premalignant lesion of the oral mucosa. We studied the colonization of Candida in oral leukoplakia using direct microscopy, culture and histopathology to determine if there is a statistical correlation between Candida invasion and the clinical appearance and presence of epithelial dysplasia in leukoplakia. Samples were collected from 40 patients with oral leukoplakia and 21 controls. The swabs collected were used to inoculate Sabouraud's dextrose agar slant and for direct microscopy with Gram's stain. Culture growths were subjected to germ tube and corn meal agar tests to differentiate between Candida albicans and non-albicans groups. Biopsies were also done in all patients for histopathological confirmation; Gomori's methanamine silver stain was used to identify fungal invasion of lesional epithelium. Nineteen cases of leukoplakia showed Candida on direct smears, compared to 3 controls. Eighteen cases and one control showed growth of Candida on culture. Non-homogenous leukoplakia showed a higher positivity rate on microscopy and culture than homogenous lesions. All these correlations were statistically significant. Forty percent of leukoplakia cases were simultaneously positive for Candida on direct microscopy, culture and histopathologic evaluation. No significant difference was found between non-dysplastic and distinctly dysplastic lesions with respect to Candida detection on microscopy or culture.

Pathology of Mycotic Tracheitis in Poultry

A case of mycotic tracheitis in an adult Rhode Island Red bird of about 20 weeks of age is described here. The bird had a history of dyspnoea, gasping and was dull prior to death. On postmortem examination lungs showed multiple circumscribed granulomatous nodules in the lungs and the trachea was occluded with caseous plugs. Microscopically there were fungal hyphae penetrating the tracheal mucosa together with a caseative plug having central necrotic mass adhering to the tracheal wall, foci of severe congestion and hemorrhage, fungal granuloma surrounded by mononuclear cell infiltration, giant cell, fungal hyphae and fibrous tissues were recorded. The fungal hyphae were also demonstrated by Grocott's methanamine silver stain.

Acquired perforating dermatosis and Addison’s disease due to disseminated histoplasmosis

Acquired perforating dermatosis (APD) is a rare disorder characterized by transepidermal elimination of contents from dermis with minimal disruption of surrounding structures, believed to be due to altered expression of dermal proteins. Its occurrence in patients with systemic mycosis has never been reported. We report a 60-y gentleman who presented with features of adrenal insufficiency (nausea vomiting, hypotension and increased pigmentation) for 4 mo, multiple hyperpigmented pruritic nodules with central keratinous plug over extensor surface of both lower limbs along with hepatosplenomegaly of one month duration. Investigations revealed low cortisol (2.3 μg/dl; normal: 5–34 μg/dl), elevated ACTH (68 pg/ml; normal: 5–15 pg/ml), enlarged bilateral adrenals with hepatosplenomegaly on CT. Methanamine silver staining of fine needle aspiration from the adrenals and bone marrow aspiration showed numerous oval yeast cells suggestive of histoplasma. Histopathology of biopsy of one of the skin nodules revealed transepidermal elimination process characterized by invagination of epidermis with extrusion of collagen bundles suggestive of APD. Patient improved with hydrocortisone replacement and there was clinical improvement with resolution of skin lesions following amphotericin-B and itraconazole therapy. This is probably the first reported case of APD in a patient with disseminated histoplasmosis who had presented with Addison’s disease.

Evaluation of PCR based DNA sequencing targeting mitochondrial larger subunit (mtLSU) region for the rapid detection of Pneumocystis jirovecii in sputum from HIV positive patients

Materials and methods A total of 104 sputum specimens from HIV positive and clinically suspected PJP patients attending Government Hospital of Thoracic Medicine during Dec 2009-Mar 2011 were included in the study. Direct staining of sputum specimens using KOH Calcoflour and Grocott methanamine silver staining (GMS) and nested PCR targeting mitochondrial larger subunit region (mtLSU) was optimized. The optimized PCR was applied on all clinical specimens and amplified products were subjected to DNA sequencing and the sequencing results were analyzed using BLAST analysis software to deduce the sequence homology.

Fungal Colonization of a Pneumonectomy Stump

A 59-year-old woman developed mild recurring hemoptysis once a week for several months after a fall with trauma to the chest. Sixteen years earlier she had undergone a right pneumonectomy at a hospital elsewhere for sequelae of pulmonary tuberculosis. Bronchoscopy, performed because of the recent hemoptysis, showed material in the pneumonectomy stump. The material had a gelatinous appearance, green color with a pale margin, and oblique striations. The material was removed by grasping with forceps and withdrawing the bronchoscope. Grocott methanamine silver stain was positive for septate, nonpigmented fungal organisms. Anatomic pathology microscopy also showed mucous, acute inflammatory cells, and necrotic tissue. Cytopathology of washings from the bronchial stump showed rare degenerated benign bronchial epithelial cells and fungal hyphae. Acid fast bacilli smears and cultures were negative. Bacterial cultures showed 3+ Pseudomonas aeruginosa. The patient had no further hemoptysis.

Fatal rhino-orbito -cerebral infection caused bySaksenaea vasiformisin an immunocompetent individual: First case report from India

A 56-year-old female patient presented with history of nasal blockage, nasal bleeding, headache, since one month. On admission the patient developed severe headache, decreased vision of eyes and blindness. Clinical diagnosis of rhino-orbital involvement was confirmed by computerized tomography of the paranasal sinuses. The diagnosis of fungal infection was confirmed by KOH examination, haematoxylin and eosin examination and Gomori's methanamine silver stain of the biopsy material. Diagnosis was confirmed by culture on Sabouraud Dextrose agar, slide culture on Czapek Dox medium and the isolate was identified as Saksenaea vasiformis. The patient was started on intravenous amphotericin B (received only one dose before succumbing), but she did not respond to the treatment, developed hemiparesis, slurred speech, diminished reflexes and ultimately died. The involvement of the brain was confirmed by computerised tomographic scan. We believe this case to be the first case of rhino-orbito-cerebral infection due to Saksenaea vasiformis and that of second case in an immunocompetent patient in the world.

Histopathologic Assessment of Fungal Involvement of the Paranasal Sinuses in Turkey

ObjectiveTo assess paranasal sinus material histopathologically for the presence of fungus.Material and MethodsParaffin-embedded archival biopsy samples of patients who underwent endonasal sinus surgery between 1992 and 2002 were retrospectively assessed for the presence of fungi. Hematoxylin-eosin-stained sections of the materials were re-evaluated, and Gomori's methanamine silver stain was also applied as required.ResultsFungus (Aspergillus) was detected histopathologically in only 21476 patients, both of whom were immunocompetent. One patient was considered to have chronic indolent sinusitis and the other allergic fungal sinusitis.ConclusionAlthough histopathologic assessment is one of the most important diagnostic tools, on its own it may lead to underestimation of fungal involvement of the paranasal sinuses. Alternatively, fungal involvement of the paranasal sinuses may be very infrequent in Turkey.

Detection of fungal organisms in eosinophilic mucin using a fluorescein-labeled chitin-specific binding protein.

The ability to identify fungal hyphae in patients with chronic rhinosinusitis (CRS) has been inconsistent. A new fluorescein-labeled staining method targets chitin found in fungal cell walls. We hypothesize that this method would be able to more consistently detect fungi within the mucin of CRS patients. Fifty-four consecutive CRS surgical patients were evaluated. After ensuring sensitivity and specificity of this new method, all specimens were stained with either fluorescein-labeled chitinase or Grocott methanamine silver stain for comparison. All 54 specimens contained eosinophilic mucin on hematoxylin and eosin staining. One or more fungal hyphae could be visualized within the mucin of 54 (100%) of 54 specimens stained using the fluorescein-labeled chitinase. Only 41 (76%) of 54 of the specimens stained with the Grocott methanamine silver stain technique demonstrated fungi. The fluorescein-labeled chitinase-staining technique has greater sensitivity in detecting fungal organisms within eosinophilic mucin. Fungal organisms are present in the mucin of CRS patients.

Occurrence ofPneumocystis carinii organisms in a peritoneal effusion from a patient with the acquired immunodeficiency syndrome

We describe a unique case of Pneumocystis carinii organisms within a peritoneal effusion of a patient with acquired immunodeficiency syndrome (AIDS). The patient, a 28-yr-old homosexual male with profound immunosuppression, presented with splenomegaly and ascites. A peritoneal tap was performed for diagnostic purposes. Cytologic examination of the cytospin specimen showed reactive mesothelial cells along with aggregates of debris reminiscent of the foamy exudates seen in the alveoli of lungs infected with the Pneumocystis carinii organism. A modified methanamine silver stain was performed and revealed the presence of scattered Pneumocystis carinii in the specimen. Extrapulmonary Pneumocystis carinii infection is occasionally seen in AIDS, but we could find no other reports of its detection in peritoneal fluid.