Gomori's Methenamine Silver Nitrate Research Articles

Modification of Grocott's staining procedure with heat treatment and oxidation by periodic acid for mucormycosis in tissue: a method to detect Mucor spp.

The sensitivity of the Grocott-modified Gomori's methenamine-silver nitrate technique for the detection of fungi is sometimes low, especially for Mucor spp. We modified the Grocott technique by replacing chromic acid with periodic acid in the oxidation step. The use of periodic acid instead of chromic acid enhanced the detectability of Mucor spp. in histopathological sections. Other parameters should be assessed with a high number of cases under different conditions. We propose our protocol as one of the options in practice, especially in cases suspected of Mucor spp. infection.

Esophageal Leukoplakia.

CASE REPORT A 64-year-old white man with a history of tobacco use and stable weight presented to the gastroenterology clinic due to an 11-month history of solid-food esophageal dysphagia. On physical examination, patient was alert and in no acute distress. His vital signs were within normal limits. His head and neck examination revealed normal oropharyngeal mucosa. No thyromegaly, lymphadenopathy, or masses in his neck were noted on palpation. Abdominal examination presented normal bowel sounds, without distension or tenderness. Upper endoscopy demonstrated white plaques at the gastroesophageal junction (Figure 1).Figure 1.: Upper endoscopy showing white plaques at the gastroesophageal junction.Biopsies obtained from this abnormality showed esophageal epidermoid metaplasia consistent with leukoplakia and mild patchy active chronic inflammation. Gomori methenamine–silver nitrate stain for fungal organisms was negative. Given the potential for malignant transformation of leukoplakia, endoscopic resection was suggested for the patient. An endoscopic submucosal dissection was planned, due to the lesion size to ensure compete removal. Chromoscopy with methylene blue was used to mark the borders of the lesion, and endoscopic submucosal dissection was accomplished without adverse events (Figure 2). Histologic examination confirmed hyperplastic squamous esophageal mucosa with marked hyperkeratosis without dysplasia and negative margins. During the 2-year follow-up, the patient reported complete resolution of his dysphagia. An upper endoscopy was ordered for surveillance.Figure 2.: Chromoscopy with methylene blue was used to mark the borders of the lesion.Epidermal metaplasia is a histological correlate to a white patch on the surface of a mucous membrane that cannot be rubbed off, known as leukoplakia.1 It is a common finding in oral mucosa, but rare in esophageal mucosa.1,2 The etiology of this condition in the esophagus is unclear and thought to be associated with esophageal tobacco and alcohol exposure or possibly an unusual response to chronic acid reflux.1,3,4 Although generally asymptomatic, symptom presentation can include dysphagia.3 Endoscopic features include slightly elevated whitish surface, which can be translucent or cobblestone-like in appearance.2,4 The natural history is unknown. As adjacent dysplasia and possible squamous cell carcinoma of the esophagus has been reported, and it is suggested that there is a potential for malignant transformation and therefore strict surveillance or endoscopic resection or ablation is warranted.1,5 DISCLOSURES Author contributions: PV Hernandez drafted the manuscript. D. Snyder, A. Kahn, KK Wang, DA Katzka, JL Horsley-Silva revised the manuscript. JL Horsley-Silva is the article guarantor. Financial disclosure: None. Previous presentation: This case report was presented as a poster at the 2019 ACG Annual Meeting; October 25-30, 2019; San Antonio, TX. Informed consent was obtained for this case report.

Rhino-orbito-cerebral infection due to Rhizopus in a diabetic patient

A 55-year-old diabetic female presented with persistent headache, bilateral nasal blockage, common cold and nasal discharge since 10 days. The patient also complained of bilateral nasal bleeding, anosmia and ptosis of the eye since the last five days. Clinical diagnosis of rhino-orbito-cerebral was confirmed by computed tomography (CT) scan of paranasal sinuses and brain. The diagnosis of fungal infection due to Rhizopus species was confirmed by Potassium hydroxide (KOH) examination, Hematoxylin and Eosin staining and Gomori's Methenamine Silver Nitrate staining on mucosal biopsy, followed by culture on Sabouraud's Dextrose agar. Surgical debridement was done and intravenous Amphotericin B was administered to the patient, who responded well to the treatment.

′Unstained′ and ′de stained′ sections in the diagnosis of chromoblastomycosis: A clinico-pathological study

Diagnosis of chromoblastomycosis is frequently missed for many reasons: (1) rarity of the lesion (2) requirement of careful search for diagnostic 'sclerotic' bodies which are often sparse in clinical material such as tissue and exudates (3) often they elicit tissue reactions such as verrucous lesion and micro abscesses, misleading the diagnosis (4) lack of 'clinical suspicion'. (1) To compare the feasibility of 'unstained', 'stained' and 'de stained' sections in identification of the diagnostic ' sclerotic' bodies (2) To study histopathological features of chromoblastomycosis, and (3) To highlight the importance of clinical suspicion in the diagnosis of chromoblastomycosis. This is a retrospective study conducted on six clinically unsuspected, histopathologically diagnosed cases of chromoblastomycosis. Most common clinical presentation was verrucous lesion over extremities affecting adult men engaged in outdoor works. Sclerotic bodies and their characteristic features were easily identified in both 'unstained' and 'de stained' sections. Special stains such as Fontana Masson and Gomori's methenamine silver nitrate are useful for demonstrating hyphal forms in keratinized layers and for illustration purposes. Both 'unstained' and 'de stained' sections can be used for rapid screening of sclerotic bodies. 'De stained' sections can be used as a suitable substitute for special stains for confirmation and for retrospective review of all verrucous lesions to diagnose the missed lesion. Clinical suspicion is very important in the diagnosis of chromoblastomycosis as it helps pathologist to screen for diagnostic sclerotic bodies.

Utility of reflex gomori methenamine silver staining forPneumocystis jirovecii on bronchoalveolar lavage cytologic specimens: A review

Pneumocystis jiroveci (Pj; formerly Pneumocystis carinii) is an opportunistic pathogen causing life-threatening pneumonia (Pneumocystis pneumonia) in immunosuppressed individuals. Its diagnosis is dependent on identification in bronchoalveolar lavage (BAL) specimens. Gomori's methenamine silver nitrate (GMS) stain has been advocated to highlight the organisms in BAL specimens. This study was performed to determine the utility of reflex GMS staining on all BAL specimens for identifying Pj.All BAL specimens from years 2000 to 2004 were processed as cytospins and stained with Papanicolaou (Pap) and GMS stains. A total of 2,984 BAL specimens were identified. A total of 116 (3.9% of total BAL) BAL specimens were diagnostic of Pj. The diagnostic specimens were grouped as follows: 103 (88.8% of total positive cases) Pj identified with both Pap and GMS staining; 11 (9.5% of total positive cases) Pj identified only with Pap staining; and 2 (1.7% of total positive cases) Pj identified only with GMS staining. In conclusion, the prevalence of Pj in BAL specimens is 3.9%, which can be attributed to improved management of immunocompromised patients. Performing reflex GMS staining on all BAL specimens does not improve the diagnostic identification of Pj since the majority (98.3%) of diagnoses can be rendered on Pap stained slides. A cost analysis for GMS staining on 2,879 GMS-negative BAL specimens was estimated at $143,950. Thus, from diagnostic and cost benefit perspectives, GMS staining can be recommended only on cases where Pap stain is negative, and the clinical presentation is consistent with Pneumocystis pneumonia.

Histological Detection of Aquatic Fungi by Uvitex 2B, a Fluorescent Dye

Conventional staining methods for fungal detection are time consuming, technically demanding, and instable dye-affinity in different fungal species, especially in aquatic fungi. In this study, Uvitex 2B [4, 4-BIS (2-di (2-hydroxyethyl) -amino-4 - (3-sulfophenylamino) -1, 3, 5-triazine-6-ylamino) -stilbene-2, 2'-disulfonic acid, sodium salt] was applied to detect fungal elements in paraffin sections of some aquatic animals. As a result, it was found that Uvitex 2B was superior to Gomori's methenamine-silver nitrate Grocott's variation, periodic acid-Schiff reaction and Schmorl's method for staining aquatic fungi in tissue sections. In addition, Uvitex 2B required much shorter time and less specialized skills in the staining procedure than Grocott. Although it has been known that oomycete fungi are difficult to be detected in histopahological sections because of their unstable stainabilities with other staining methods, Uvitex 2B provided excellent results to detect them in tissues of aquatic animals.

Adiaspiromycosis

Adiaspiromycosis (ad"i-ah-spi"ro-mi-kósis) is a worldwide, noninfectious, nonarthropod transmitted fungal infection of lower vertebrates, most commonly rodents. Humans become an accidental host by inhaling dust-borne spores (conidia) of the saprophytic soil fungus, Emmonsia crescens (recently renamed Chrysosporium parvum variety crescens). We report 11 cases of this unusual deep mycosis from South America, Europe, and the United States. The severity of the disease depends on the number of spores inhaled. In limited inoculum, the disease remains localized (two cases), whereas in heavy inocula the fungus involves both lungs (nine cases) and presents as a diffuse reticulonodular infiltrate. In this disseminated form, patients usually complain of cough, dyspnea on exertion, and low-grade fever mimicking other systemic fungal infections and tuberculosis. It is difficult to unmask the fungus because it is not easily cultured nor is there a reliable serologic test. Therefore, a biopsy is required and the pathologist must recognize the large (ranging in size from 50 to 500 microns), round, Gomori methenamine-silver nitrate and periodic acid-Schiff reagent-positive spherules with a trilaminar wall. The spherules can be surrounded by either suppuration, epithelioid granulomas with or without necrosis, or concentric, hyalinized fibrosis. In the latter chronic stage, the organism may collapse, forming a variety of sizes and shapes thereby resembling other fungi, helminths, mineral particles, or inhaled pollen grains. Clinically, the infection most commonly regresses spontaneously, but may persist, or rarely progress, requiring surgical intervention with limited resection to attain cure.

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.

Pneumocystis carinii Infection in Corticosteroid-Treated Cats

Corticosteroids were administered to produce Pneumocystis carinii infection in cats. Six of 10 cats, injected intramuscularly for 97-141 days with 2 mg/cat twice weekly of betamethasone sodium phosphate, developed a light infection with P. carinii. Six of 7 cats, injected intramuscularly for 11-168 days with 10-25 mg/cat weekly of prednisolone acetate, also developed a light infection with P. carinii. There was no significant difference in the infection rate between the sexes and ages of the cats. Using Giemsa staining and Gomori's methenamine silver nitrate stain, P. carinii organisms were indistinguishable morphologically from human and rat P. carinii. The cysts and trophozoites were usually present singly or in small groups, and they always were adhering to the periphery of alveoli. The inflammatory changes were inconspicuous except for the fact that alveolar macrophages often were seen. Corticosteroid-treated cats should be useful in the study of experimental P. carinii infection. This is the first reported case of experimentally induced P. carinii infection in cats.

Diagnosis of Pneumocystis carinii in sputum liquefied with the mucolytic agent dithiothreitol (sputasol).

1. Light microscopic investigations : Sputasol treatment did not affect the staining properties of either the cyst wall or the parentheses-like structures by Gomori's methenamine silver nitrate (GMS) stain. Detection of Pneumocystis carinii was easier in the Sputasol-treated preparations because it was possible to prepare thin smears of the sediment, and because the cysts were concentrated. The amount of time needed for microscopic examination was reduced to the decreased amount of sediment in the Sputasol-treated samples. 2. Transmission electron microscope investigations : In the ultrastructure of P. carinii cysts stained with GMS stain, the heavier deposition of silver particles on the internally thickened parts of the cyst wall corresponded exactly with the distinctive appearance of the parentheses-like structures seen in light microscopic observations.

Localization of silver deposits on Pneumocystis carinii treated with Gomori's methenamine silver nitrate stain

The question which portion of the pellicle of Pneumocystis carinii is actually stained by the Gomori's methenamine silver nitrate (GMS) method was solved with the aid of electron microscopy. Silver particles were specifically deposited on the electron-lucent middle layer of the precyst, cyst and ruptured cyst, whereas such deposition was not found on the pellicle of the trophozoite, probably due to the absence of the electron-lucent middle layer in the trophozoite. These deposits increased in proportion to the duration of staining and to the thickness of the lucent middle layer. Therefore, the lighter colored organisms as observed under the light microscope are considered to be the precysts because they have thinner GMS-positive middle layers than those of cysts. So-called parentheses-like structures or rings are one of the most characteristic light microscopic features of P. carinii cysts when stained with GMS. The present study indicates that these structures must be of cyst wall origin rather than of cytoplasm origin, because the thickened part of the cyst wall showed a dense deposition of silver particles, probably corresponding to the parentheses-like structure. On the other hand we also observed, in the endogeny form of trophozoite, a thin GMS-positive layer in the parent pellicle though this pellicle showed no differences from those of daughter and common trophozoites when observed by means of ultrathin section microscopy.

Pneumocystis infection in macaque monkeys: Macaca fuscata fuscata and Macaca fascicularis.

Retrospective examination of lungs from 128 monkey necropsies was attempted for Pneumocystis infection using special strains, including toluidine blue-O and Gomori's methenamine silver nitrate. Four Japanese monkeys (7.7%), Macaca fuscata fuscata, and one crab-eating monkey (7.7%), Macaca fascicularis, were found to have Pneumocystis infection. The organism was found in young and infant animals. At the time of death, one infant and two young monkeys were debilitated and/or emaciated. Pneumocystis infection was considered an important lesion which could have caused reduced respiratory function in two of the Japanese monkeys, but constituted only an incidental finding in the others.

Light microscopic observation of the socalled parenthesis-like structure of Pneumocystis carinii cysts in smears stained by Gomori's methenamine silver nitrate.

Light microscopic observation of the socalled parenthesis-like structure of Pneumocystis carinii cysts in smears stained by Gomori's methenamine silver nitrate.

Simultaneous demonstration of cyst walls and intracystic bodies of Pneumocystis carinii in paraffin embedded lung sections using Gomori's methenamine silver nitrate and Giemsa stain.

Simultaneous demonstration of cyst walls and intracystic bodies of Pneumocystis carinii in paraffin embedded lung sections using Gomori's methenamine silver nitrate and Giemsa stain.

Morphology, development and behavior of pneumocystis carinii observed by light-microscopy in nude mice

The present paper describes the morphology, development and behavior of Pneumocystis carinii, especially of the trophozoites in the alveoli of conventionally raised BALB/c nude mice, by using phase-contrast microscopy, paraffin sections and semiultrathin sections embedded in JB-4 plastic. Under phase-contrast microscopy, trophozoites were ameboid in external appearance and 2 to 8 micron in diameter. Usually they have one or more lucid spherical vacuoles and one less lucid nucleus in the cytoplasm. Maturation and independence of intracystic bodies were observed in the developing cysts. The intracystic bodies were polymorphic, i.e. spherical, ameboid or elongated. The paraffin sections using the double staining of P. carinii with Gomori's methenamine silver nitrate (GMS) and Giemsa, showed a small number of mature cysts containing intracystic bodies and a large number of trophozoites within the characteristic honeycombed material in the alveolar spaces. In order to investigate the morphology and parasitizing behavior of the trophozoites and cysts as well as the response of alveolar tissue in more detail, semiultrathin sections of 0.5-2 micron thickness were made from the materials embedded in JB-4 plastic and stained with Giemsa. In lightly infected alveoli, some trophozoites and cysts were found to be closely attached to the Type I alveolar epithelial cells. In heavily infected alveoli, almost all alveoli were filled with trophozoites, cysts, debris of the host cells, and occasionally phagocytic cells (macrophages and neutrophils) containing cysts or trophozoites were found. It was noted that the cysts were very few in number (1%) compared with the number of the trophozoites in nude mice in a 0.4 mm2 area of 2 micron thick lung sections. The host tissue of nude mice in this study was not as strongly affected by the organisms as that in cortisone-treated rats and P. carinii pneumonia patients. In the present study the morphology of P. carinii found in nude mice was not different from that found in rats and in man.

Primary pulmonary sporotrichosis

Pulmonary sporotrichosis in the absence of lymphocutaneous disease is unusual; however, its incidence may be greater than previously recognized. This report describes the AFIP experience in eight cases of primary pulmonary sporotrichosis and reviews an additional 23 cases. The disease is often a bilateral, apical, chronic and cavitary, progressive, destructive, and debilitating infection, most often seen in middle-aged men with a history of alcoholism and chronic obstructive pulmonary disease. In this clinical setting, pulmonary sporotrichosis closely mimics tuberculosis or histoplasmosis. However, this clinical charade can be unmasked by serologic tests, cultures, and identification of the causative agent, Sporothrix schenckii, in sections of paraffin-embedded lung containing necrotizing granulomas and stained with periodic acid-Schiff and Gomori methenamine-silver nitrate. Previously not recognized is the presentation of primary pulmonary sporotrichosis as a solitary, peripheral, necrotizing pulmonary nodule, observed in two patients. Chronic cavitary pulmonary sporotrichosis is usually refractory to drug therapy; however, when combined with surgical resection, cure can be attained.

Pneumocystis carinii pneumonia. An approach to rapid laboratory diagnosis.

Laboratory procedures used to establish the diagnosis of Pneumocystis carinii pneumonia were evaluated using an experimental murine model. Touch preparations and suspension smears were prepared from lung tissue know to contain Pneumocystis cysts. These preparations were stained by a variety of methods known to demonstrate either cyst forms or sporozoites and trophozoites. Suspension smears proved to be superior to touch preparations in terms of cyst content and homogeneity of staining. Also, methods that stain cyst forms were superior to those that stain sporozoites and trophozoites for location and identification of organisms. The authors believe that suspension smears prepared from lung tissue and stained with toluidine blue O should be examined initially as a rapid screening method for Pneumocystis cysts. When the results of this initial screen are negative or inconclusive, additional suspension smears stainded by the modified Gomori methenamine silver nitrate method should be examined, pending availability of histologic sections.

Histochemical observations on Pneumocystis carinii: selective demonstration of honeycomb forms.

Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid--sodium bisulfite--resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.

Percutaneous transthoracic needle aspiration of the lung. Diagnosing Pneumocystis carinii pneumonitis.

Percutaneous transthoracic needle aspiration was performed on 228 occasions to obtain lung specimens from 202 patients with suspected Pneumocystis carinii pneumonitis. In 121 patients the diagnosis was established by identifying P carinii organisms in lung aspirates. Six patients whose aspirates did not contain P carinii were found to have the organism at autopsy. Findings from toluidine blue O and Gomori methenamine silver nitrate stains were equally satisfactory for detecting P carinii, but the percentage of specific diagnosis was higher when specimens were stained with both. Pneumothorax that required a thoracotomy tube occurred in 39 patients. Other infectious agents, either bacteria or fungi, were found in only four patients. Percutaneous pulmonary needle aspiration--when performed under fluoroscopic guidance--is a rapid and effective method for the diagnosis of P carinii pneumonitis.

Mycotic abortion

History and Clinical Signs : Abortions ordinarily are sporadic and the total incidence within a herd seldom reaches 10% of the pregnant cows. January through March is generally the period of highest incidence in the northern hemisphere. There are no common prodromal or postabortion signs in the cow. Gross Lesions : Severe placentitis is a constant lesion with necrosis, thickening and cupping of the cotyledons, leathery thickening of the intercotyledonary tissue and adventitious placentation occurring commonly. In about 25% of the cases ringworm-like lesions of the fetal skin occur. The fetal tissues usually do not have signs of preexpulsion autolysis. Frequently the lungs are partially expanded. The carcass may be somewhat dehydrated and emaciated. Microscopic Lesions : Necrotizing inflammation of the chorionic villi often with vasculitis and thrombosis is a constant lesion. Varying amounts of mycelia and necrotic intervillus tissue are associated with this lesion. Occasionally suppurative bronchopneumonia with associated mycelia occurs. Mycotic skin lesions of epidermitis and folliculitis associated with mycelia occur in approximately 25% of mycotic abortion cases. Cultural Procedures : The etiologic fungus usually can be isolated from the fetal abomasal contents, the placenta, occasionally from the fetal lungs, and when skin lesions are present, from the skin. In some cases, mycotic lesions are present but the organism cannot be isolated. The ubiquity of fungi must be kept in mind when interpreting the results of culture procedures. Serologic Procedures : No serologic procedures are commonly used. Special Procedures : The use of special stains, such as Gridley's, Gomori's methenamine silver nitrate and PAS, aid greatly in determining the presence of mycelia in tissues. Microscopic examination for mycelia of KOH digested placenta or skin scrapings aids in providing an early tentative diagnosis. Preferred Diagnostic Procedures : 1. 1. Demonstrate gross or microscopic lesions of placentitis with associated mycelia. 2. 2. Demonstrate skin lesions with associated mycelia. 3. 3. Demonstrate bronchopneumonia with associated mycelia. 4. 4. Culture of fungi from fetal abomasal content, skin lesions or lungs.