Grocott's Methenamine Silver Stain Research Articles

Infectious Granulomatous Dermatitis Associated With Rothia mucilaginosa Bacteremia: A Case Report

Infections with rare pathogens are being recognized with increasing frequency in severely immunocompromised patients. As a result of these patients' underlying compromised defenses and susceptibility to atypical organisms, tissue biopsies from patients within this population may demonstrate nonclassical histopathological findings. Here, we describe an unusual granulomatous reaction to gram-positive cocci in the skin of a 52-year-old man undergoing salvage chemotherapy for acute myeloid leukemia. The patient presented with a papular eruption on the arms, trunk, and face and fever; concomitant blood cultures were positive for Rothia mucilaginosa and Streptococcus salivarius. Histologic evaluation revealed a granulomatous dermatitis associated with numerous small, round, predominantly intracellular bacteria. Classically, cutaneous infiltrates associated with coccoid bacterial infections are suppurative and not granulomatous. The intracellular organisms stained positive for Gram, periodic acid-Schiff, and Grocott methenamine silver stains, suggestive of R. mucilaginosa. Rothia mucilaginosa, a component of the oral flora, was first reported as a human pathogen in 1978. Although the majority of cases in the literature have described R. mucilaginosa bacteremia, other reported manifestations include meningitis, endocarditis, pneumonia, osteomyelitis, and peritonitis. To our knowledge, however, only 1 prior report has described a cutaneous manifestation of R. mucilaginosa septicemia, which occurred in a patient with neutropenia. This is the second reported case of an infectious granulomatous dermatitis associated with R. mucilaginosa bacteremia and raises awareness of this unusual histopathological presentation in the setting of a bacterial infection affecting the skin.

Prosthetic valve endocarditis: clinicopathological correlates in 122 surgical specimens from 116 patients (1985–2004)

Few studies have documented the clinicopathological features of prosthetic valve endocarditis independently of native valve endocarditis. Retrospective analysis of patients undergoing cardiac surgery for prosthetic valve endocarditis at our institution (1985-2004). Medical records and microscopic slides were reviewed from 116 patients for demographics, infecting organisms, comorbidities, and pathologic features. Patients were 12-86 years old (mean, 59 years). Among 122 valves, 64% were from men and 67% were purely regurgitant. Aortic prosthetic valve endocarditis frequently affected men (76%); mitral prosthetic valve endocarditis often affected women (62%). Embolization occurred in 35% and heart failure in 32%. Prevalent predisposing conditions were the prosthetic valve alone (43%) and diabetes mellitus (20%). Prosthetic valve endocarditis was aortic or mitral in 98% and was active in 70%. Annular abscess or paravalvular leak affected mechanical valves more frequently than bioprosthetic (89% vs. 65%; P=.001). Causative organisms (n=116) included Staphylococcus aureus (30%), coagulase-negative staphylococcus (22%), viridans streptococci (18%), enterococci (10%), other streptococci (8%), and other organisms (12%). S. aureus was the most prevalent cause of early-onset (38%) and late-onset (30%) prosthetic valve endocarditis. Coagulase-negative staphylococcus caused early-onset (31%) and most intermediate-onset (40%) disease and had a shorter median implantation-to-infection time than other organisms (6.5 vs. 61.3 months; P<.001). Viridans streptococci and enterococci primarily caused late-onset endocarditis. For active infections by cocci, most cases exhibited strong Gram staining, but four showed only strong Grocott methenamine silver staining. Cocci accounted for 83% of infections. Early-onset prosthetic valve endocarditis was primarily staphylococcal, and late-onset prosthetic valve endocarditis resembled native valve endocarditis. Both Gram and Grocott methenamine silver stains were necessary to reliably identify organisms microscopically.

In Situ Hybridization for the Differentiation of Actinomyces and Nocardia in Tissue Sections

The specific identification of filamentous bacteria in tissue sections can be difficult. The filamentous bacteria Actinomyces and Nocardia often produce similar host responses and single bacterial organisms seem morphologically similar; however, their differentiation may be clinically significant. In situ hybridization (ISH) may assist in the rapid and accurate identification of these microorganisms. In this study, DNA probes were directed against the variable regions of 16S ribosomal RNA genes of multiple Actinomyces and Nocardia spp. Probes were tested on 26 formalin-fixed, paraffin-embedded tissue specimens, each of which contained diagnostic foci of filamentous bacteria confirmed by both Gram and Grocott methenamine silver stains. On the basis of histology and clinical features, cases were classified as Actinomyces-related, n = 13 with 6 culture-proven cases and Nocardia-related, n = 13 with 11 culture-proven cases. Using this classification, all cases were assessed for cross-reactivity using other species-specific probes and probe specificity was determined. Overall, Gram and Grocott methenamine silver histochemical stains (100% sensitivity) were more sensitive than ISH (77% sensitivity for both Actinomyces and Nocardia probes). The slender caliber of filamentous bacteria was a limitation for ISH interpretation and necessitated careful examination of some slides. Probes demonstrated 100% specificity for identifying both species, 100% positive predictive value and 81% negative predictive value. No mixed infections were observed. This study demonstrates that ISH is highly specific for distinguishing between Actinomyces and Nocardia spp. in tissue sections. Although histochemical stains demonstrate greater sensitivity for organism detection, ISH is a rapid and specific technique that is especially useful for evaluating culture-negative or clinically unsuspected cases of filamentous bacterial infection.

Screening Pneumocystis carinii pneumonia in non–HIV-infected immunocompromised patients using polymerase chain reaction

We evaluated the clinical value of polymerase chain reaction (PCR) for screening Pneumocystis carinii pneumonia (PCP) in non–HIV-infected immunocompromised patients. We retrospectively analyzed PCR and Grocott's methenamine silver (GMS) staining on 1044 clinical specimens obtained from 756 patients. Positive rates of PCR and GMS staining in sputum specimens were 21.1% and 9.5%, respectively ( P < 0.01), and in bronchoalveolar lavage (BAL), specimens were 31.9% and 25.5%, respectively. Among 5 patient groups, the highest GMS staining and PCR positive rates were observed in immunosuppressed patients. In 28 GMS staining-positive patients, positive rates of PCR and GMS staining after a short anti-PCP treatment were 39.3% and 17.9%, respectively. In 5 patients with positive PCR but negative GMS staining results in initial sputum examination, GMS staining result turned positive in subsequent BAL specimens. In conclusion, PCR is sensitive for detection of Pneumocystis in sputum specimens and is useful for screening PCP in non–HIV-infected high-risk patients.

Cutaneous plasma cell granuloma: report of a case with novel histologic and immunohistochemical findings

A 50-year-old man presented with an asymptomatic, 1.5 x 1.5 cm, dark-brown noduloplaque with a rubbery consistency (Fig. 1) on the lateral aspect of the left lower leg of uncertain duration. His general condition was healthy, and he did not recall any trauma or insect bite at this site. No similar skin lesions were found elsewhere and no lymphadenopathy was observed. The lesion revealed a nonencapsulated, but well-circumscribed, deep dermal nodule with several lymphoid aggregates and germinal center-like structures within the tumor and also at the periphery, when examined microscopically at scanning power (Fig. 2a). The epidermis showed no remarkable changes, except for basal hyperpigmentation. At higher power, a mixed inflammatory infiltrate composed of histiocytes, foamy histiocytes (Fig. 2b), lymphocytes, and abundant plasma cells (Fig. 2c) with Russell bodies was revealed. The stroma contained mainly hyalinized and sclerotic collagen fibers (Fig. 2d). Prominent venules were noted, especially in the sclerotic areas, and some were surrounded by dense collagen fibers. No vasculitis or emperipolesis was found. No foreign materials were observed by polarization microscopy, and no organisms could be identified by periodic acid-Schiff (PAS), Grocott methenamine silver (GMS), Giemsa, Gram, acid-fast, or fite stains. The results of testing for infection by Epstein-Barr virus (EBV) (latent membrane protein 1, LMP-1) were negative. No spindle cells were found in the lesion. Immunohistochemical studies demonstrated mature plasma cells stained with CD138, and polyclonality was confirmed by the expression of both kappa and lambda light chains. The germinal center-like lymphoid aggregates were found to be B cells, which reacted positively with CD20. Scarce S100-positive cells and even rarer CD1a-positive cells were detected. Test results for smooth muscle actin (SMA) and anaplastic lymphoma kinase (ALK) were negative. Abundant CD68+ macrophages were observed within the lesion (Fig. 3a), and about 50-75% of the inflammatory cells were found to express cyclooxygenase-2 (COX-2) (Fig. 3b). The patient's condition was diagnosed as cutaneous plasma cell granuloma (CPCG). One year after excision, no evidence of recurrence was observed.

Surto de pitiose cutânea em bovinos

Setenta e seis bovinos, sem raça definida, jovens, de ambos os sexos, apresentaram lesões cutâneas multifocais nodulares, ulceradas e crostosas nas faces medial e lateral dos membros anteriores e posteriores, região ventral do pescoço, esterno e cauda. A doença ocorreu no verão e as lesões foram observadas nas regiões do corpo que ficavam grande tempo em contato com a água em canais de irrigação. Histologicamente observaram-se múltiplos granulomas e piogranulomas contendo escassas imagens negativas de hifas na área central, as quais foram melhor evidenciadas através da técnica de metenamina nitrato de prata de Grocott. O diagnóstico etiológico definitivo foi baseado na técnica de imuno-histoquímica com anticorpo policlonal anti-Pythium insidiosum. Adicionalmente, foi realizado o teste de ELISA indireto. Surtos de pitiose cutânea bovina são incomuns e, particularmente neste relato, todos os animais afetados tiveram cura espontânea das lesões dentro de duas a três semanas.

Significance of MUC5B antibody in differential diagnosis between Aspergillus species and Mucorales of fungal sinusitis

To differentiate between Aspergillus species and Mucorales of fungal sinusitis by immunohistochemistry. Formalin-fixed paraffin-embedded tissue blocks of 66 cases of fungal sinusitis were retrieved from the archival files of Department of Pathology of Beijing Tongren Hospital during the period from 2001 to 2006. The samples included 29 cases of fungal balls, 12 cases of allergic fungal sinusitis, 24 cases of chronic invasive fungal sinusitis and 1 case of acute invasive fungal sinusitis. The types of fungi were 44 Aspergillus species (31 cases of A. fumigatus, 7 cases of A. flavus and 6 cases of A. terreus) and 22 Mucorales (14 cases of Mucor species and 8 cases of Rhizopus species). Immunohistochemistry was performed with MUC2, MUC5AC and MUC5B antibodies. The results were compared with histochemical study for periodic acid-Schiff (PAS) and Grocott methenamine silver (GMS) stains. Immunohistochemical study for MUC5B showed that the positive rate of Aspergillus species was 90.9%, in contrast to 4.5% in Mucorales (P < 0.001). The expression of MUC2 and MUC5AC was completely negative, whereas PAS and GMS stains were positive in all cases. MUC5B antibody appears to be a useful immunohistochemical marker for identifying fungal types in tissue sections, especially in distinguishing between Aspergillus species and Mucorales in fungal sinusitis.

Conidiobolomycosis in Sheep in Brazil

Conidiobolomycosis is reported in the state of Piauí, in the semiarid region of northeastern Brazil. Affected sheep had depression, weight loss, serous or mucohemorrhagic nasal discharge, and cranium-facial asymmetry from exophthalmos of 1 eye, generally with increased volume of the eyeball, keratitis, and corneal ulceration. At necropsy of 60 sheep, friable masses were observed in the posterior region of the nasal cavity, often destroying the ethmoturbinate bones. Frequently, the lesions invaded the nasal sinuses, cribiform plate, orbit, and brain. The masses were irregular, granular with moist surfaces, and soft and friable with white, yellow, or tan coloration. Dissemination of the lesion to lungs was observed in 27 sheep, to the brain in 26, to lymph nodes in 3, to the kidney in 2, and to the gallbladder and heart in 1. The microscopic examination showed granulomatous inflammation composed of central necrosis surrounded by lymphocytes, epithelioid and giant cells, and fibrous tissue. In all lesions, negatively stained structures representing hyphae were surrounded by Splendore-Hoeppli material. Coagulative necrosis, thrombosis, and vasculitis were also observed. Grocott methenamine silver stain showed 8-30-microm-thick hyphae, rarely septate or ramified, irregular in shape, and with black contoured wall, sometimes with bulbous dilatation in the extremities. On electron microscopy, the hyphae had a thick double wall surrounded by cellular remnants and an inflammatory exudate. Conidiobolus coronatus was isolated from the lesions of 6 sheep. Conidiobolomycosis is an important disease of sheep in the state of Piauí, and other regions of northeastern Brazil.

The pro-apoptotic human BH3-only peptide harakiri is expressed in cryptococcus-infected perivascular macrophages in HIV-1 encephalitis patients

In the central nervous system (CNS), HIV-1 targets mainly microglia/macrophages. Like the CD4+ T cell depletion and neuronal loss in AIDS, apoptosis is thought to be involved in eliminating infected macrophages. In this study, we examined the expression of the pro-apoptotic BH3-peptide harakiri (Hrk) in brain tissues of AIDS patients. Immunoreactivity against Hrk was positive in perivascular macrophages infiltrated into some restricted lesions. Most of these immunopositive cells contained small inclusions positive for Grocott's methenamine silver staining. Confocal laser microscopy demonstrated that Hrk expression coincided with immunoreactivities against HIV-1 and Cryptococcus neoformans. Expression of Hrk mRNA was demonstrated in these cells by in situ hybridization, which indicated that Hrk is not phagocytosed material. Some pro-apoptotic bcl-family members, including Hrk, may contribute to the delayed hypersensitive reaction in AIDS, in macrophages eliminating opportunistic infection.

Invasive infection with Trichosporon inkin in 2 siblings with chronic granulomatous disease

A 9-year-old girl with autosomal recessive chronic granulomatous disease (CGD) presented with asymptomatic bilateral pulmonary infiltrates on routine computed tomography. Fine-needle aspirate of the infiltrates was obtained and showed fungal cells resembling Trichosporon inkin . The specimen grew in culture, and testing by means of both API 20C and PCR amplification confirmed the diagnosis of T inkin . The infiltrates increased in size, despite sequential therapy with voriconazole, liposomal amphotericin B, caspofungin, and posaconazole. The patient required resection of the infected lung tissue, after which she recovered completely. While she was undergoing therapy, her 13-year-old brother, also with CGD, was given a diagnosis of bilateral T inkin -induced pulmonary infection. He also required bilateral pulmonary resection for cure. These cases demonstrate the predisposition of patients with CGD to have invasive infections with unusual fungal organisms, such as T inkin . They also illustrate the difficulty of treating invasive T inkin infections with antifungal agents alone. There are 9 previously reported cases of invasive infections caused by T inkin , 3 of which are in patients with CGD. All patients required removal of infected prosthetic devices or surgical resection of infected tissue for cure.

Aborto por Aspergillus fumigatus e A. niger em bovinos no sul do Brasil

As infecções micóticas apresentam distribuição mundial e podem causar placentite e aborto em diversas espécies de animais. Entre setembro 2001 e novembro 2002, foram processados no Setor de Patologia Veterinária, Universidade Federal do Rio Grande do Sul, 147 fetos bovinos abortados com o objetivo de avaliar as principais causas de aborto infeccioso bovino no sul do Brasil. Destes, 34 estavam acompanhados da placenta. Aborto micótico foi diagnosticado em cinco casos (3,4%) mediante cultivo puro de quatro amostras de Aspergillus fumigatus e uma de A. niger associado a lesões histológicas características de fungo. Os exames virológico, bacteriológico e imunofluorescência direta para Leptospira sp foram negativos nestes casos. A idade dos fetos variava entre 5 e 8 meses de idade. Lesões macroscópicas foram observadas em quatro casos e eram caracterizadas por áreas circulares multifocais branco-acinzentadas na pele, principalmente na região da cabeça e dorso, em dois fetos, lesões nodulares no fígado em um caso e espessamento dos cotilédones em duas placentas enviadas juntamente com os fetos. Lesões histológicas foram observadas principalmente no fígado, pulmão e placenta, caracterizadas por hepatite necrótica multifocal, broncopneumonia supurativa e placentite necrótica. Através da coloração de Grocott hifas septadas foram observadas em duas placentas e nas bordas das lesões necróticas no fígado de um feto. Em dois casos hifas foram observadas somente na placenta e não no feto, salientando-se a importância deste tecido para o diagnóstico de aborto micótico bovino.

A Combined Hematoxylin and EosinIMethnamine Silver Stain for the Histological Diagnosis of Fungi in Tissue Sections

Most histochemical stains available for detection of fungi in tissue sections accentuate the fungi but do not selectively stain surrounding tissue. Therefore, the exact location of organisms in tissue and their relationship to important histological features cannot be ascertained with a routine Grocott methenamine silver stain (GMS). We evaluated the utility of a combined H&E/methenamine silver (H&E/MS) protocol that stains surrounding tissue as well as the fungi and modified it for greater rapidity, ease of performance, and improved clarity. Twenty cases of various mycotic diseases were stained with routine H&E, GMS, and the modified combined H&E/MS stain. Both the combination H&E/ MS stain and routine GMS readily demonstrated all types of fungi. However, the surrounding tissue that does not selectively stain with routine GMS was easily visualized using the combination stain, allowing a more comprehensive histopathological assessment on a single section. The histological detail was cornparable to routine H&E alone. The modified H&E/MS is quick, relatively inexpensive, and uses reagents routinely stocked by histology laboratories. This stain is a valuable addition to the pathologist's armamentarium for detecting fungi in tissue sections. (The J Histotechnol 23:341, 2000)

Cutaneous and paranasal aspergillosis in an immunocompetent patient

A 26-year-old Libyan woman presented with asymptomatic nodulo-ulcerative skin lesions present for 1 year. Three years prior to presentation, she had experienced a nasal discharge followed by the development of a nodule in the nasal cavity and a plaque on the hard palate. These lesions had gradually increased in size and ulcerated, resulting in perforation of the nasal septum and palate. Two years later, the patient noticed the appearance of skin lesions: a nodule on the right thumb and numerous nodulo-ulcerative lesions on the extremities. General physical examination was normal with no significant lymphadenopathy. Examination of the oral cavity revealed perforation of the distal nasal septum, with a perforated nodular plaque involving the entire palate, associated with subluxation of the upper incisors (Fig. 1a). On skin examination, multiple firm nodules and nodulo-ulcerative lesions with a central eschar and raised margins were observed. The lesions ranged in size from 0.5 to 5 cm and were distributed on the right hand and fingers, left upper arm (Fig. 1b), left calf, and right thigh. Routine laboratory investigations (liver function tests, serum calcium, electrolytes, lipid profile, urine and stool culture studies) were normal. Immunoelectrophoresis disclosed normal levels of immunoglobulins IgG, IgA, and IgM. Serologic studies for human immunodeficiency virus (HIV) and syphilis, and a tuberculin test, were all negative. A Giemsa-stained tissue smear was negative for Leishmania tropica organisms. Radiological studies disclosed a slight haziness of the maxillary sinuses with perforation of the nasal septum. A chest X-ray was normal. Histopathologic examination of biopsies taken from both the palate and from ulcerated and nonulcerated skin lesions was performed, and all showed similar findings. The biopsy of a nonulcerated skin lesion showed pseudoepitheliomatous epidermal hyperplasia with neutrophilic microabscesses (Fig. 2a). A dermal diffuse and nodular granulomatous mixed infiltrate of lymphocytes, histiocytes, giant cells, numerous eosinophils, and neutrophilic microabscesses was seen in all tissues examined. Septate hyphae were present both within giant cells and free in the dermis (Fig. 2b). The hyphae were branching at a 45 degrees angle and were positive on periodic acid-Schiff and Grocott methenamine silver stains (Fig. 2c). Fungal culture studies of material taken from an ulcerated skin lesion grew Aspergillus flavus. Blood cultures were negative for Aspergillus sp. or other microorganisms. The patient was treated with intravenous amphotericin B, but the medication was discontinued due to her intolerance to the drug. She was subsequently lost to follow-up.

Bacillary angiomatosis: presentation of six patients, some with unusual features

Bacillary angiomatosis (BA) is an unusual systemic vascular proliferation seen predominantly in patients with the acquired immunodeficiency syndrome. These vascular lesions are probably due to infection with a Bartonella species, most often B. henselae and, in some patients, B. quintana. BA is treatable and often curable, but without therapy, may be life-threatening. Clinically, the lesions, when superficial, are said to often resemble pyogenic granulomas, appearing polypoid histologically with an epidermal collarette. We now report six patients, three of whom showed lesions of BA morphologically and histologically distinct from the other patients reported to date. Two patients lesions appeared clinically as violaceous plaques and tumours resembling Kaposi's sarcoma; one of them had lesions histologically reminiscent of a papular angiokeratoma; and the other had lesions histologically suggestive of a combination of Kaposi's sarcoma and BA. Another patient presented with soft subcutaneous nodules which histologically showed extensive acute inflammation characteristic of an acute abscess, but which also displayed proliferating dilated small blood vessels with bulbous endothelial cells adjacent to numerous bacteria and also containing them. The Grocott-methenamine silver stain and the Warthin-Starry stain showed the organisms to better advantage in lesions of all six patients, although bacteria were also evident with the haematoxylin and eosin, periodic acid-Schiff and alcian blue stains.

Detection of Pneumocystis carinii by in situ hybridization in the lungs of immunosuppressed rats.

In situ hybridization was performed to detect rat Pneumocystis carinii in the lung sections. Rats were immunosuppressed by weekly subcutaneous injection of 10 mg/kg methylprednisolone. On the 6th, 8th and 9th week of immunosuppression, the lungs were removed and fixed in 10% neutral formalin. A 22 base oligonucleotide probe complementary to P. carinii 5S ribosomal RNA was commercially synthesized and its 3' terminal was labeled with biotin. In situ hybridization was performed utilizing manual capillary action technology on the Microprobe system. P. carinii were detected along the luminal surface of alveolar pneumocytes, in exudate of alveolar cavities, and also in secretory material of bronchioles. In the 6th week group, positive reaction was observed focally in the peripheral region of the lung sections, but the reaction was observed diffusely in the 8th or 9th week groups. In comparison with Grocott's methenamine silver stain, in situ hybridization technique can detect the organism rapidly, and can detect trophic forms very well. Furthermore, no nonspecific reaction with other pathogenic fungi and protozoa was recognized. Therefore, in situ hybridization can be a good technique to detect P. carinii in the lungs of infected rats.

Staining Method for Detecting Concurrent Viral Infections in Animals withPneumocystis Carinii

A method to identify concurrent viral respiratory infections in animals with Pneumocystis Carinii (PC) has been developed by combining immunohistochemical and conventional special staining methods. In an effort to reduce both the number of slides to be screened microscopically and to demonstrate viral expression and PC cysts in the same paraffin section, a streptavidin immunohistochemistry protocol was combined with a rapid Grocott methenamine silver stain. This method is within the expertise and technical capabilities of most research and clinical laboratories. (The J Histotechnol 17:349, 1994)

Detection of human cytomegalovirus infection in paraffin sections by modified Steiner silver stain for LM and TEM

Human cytomegalovirus (HCMV) infection is encountered more frequently in surgical specimens due to the increased number of immunosuppressed patients (organ transplants or HIV positive). Previous studies showed that HCMV infected cells in paraffin sections can be precisely labelled by in situ hybridization or by Grocott's methenamine silver stain for LM and subsequently confirmed by TEM. However, further studies indicate HCMV infection can also be identified by modified Steiner silver stain3 but in a different manner.Modified Steiner silver stained paraffin sections can be processed for TEM as follows: Coverslips are removed by immersion in xylene. A few drops of 1% OsO4, freshly dissolved in xylene, is then pipetted onto each slide for 5 minutes. Aqueous OsO4 can not be used as it will destain the sections instantly. Slides are rinsed in xylene, xylene/acetone and then 100% acetone. The sections are infiltrated with epoxy resin/acetone, embedded in epoxy resin, polymerized and subsequently separated from slides by heat as previously described.

Pulmonary Calcification Caused by Pneumocystis carinii Pneumonia

Pulmonary microcalcifications were identified in 13 patients with Pneumocystis carinii pneumonia (PCP) and acquired immune deficiency syndrome (AIDS). Several patterns of calcification were noted including bubbly, plate-like, elongate, and conchoidal forms. All calcifications contained P. carinii cysts within them as demonstrated by Grocott's methenamine silver stain. In eight of the 13 cases, the typical intra-alveolar exudate of P. carinii was absent. Therefore, the calcifications were the only histopathologic indication of prior P. carinii infection. The above-mentioned calcifications are important because in five cases they occurred without a prior diagnosis of PCP, subsequent therapy, or prophylaxis. This indicates that in some patients there is destruction of P. carinii organisms with subsequent calcification, and this can occur without therapy.

Cytology of treated and minimal Pneumocystis carinii pneumonia and a pitfall of the grocott methenamine silver stain

To clarify the role of foamy alveolar casts (FACs) in the diagnosis of Pneumocystis carinii pneumonia (PCP), we retrospectively reviewed Papanicolaou (Pap)- and Grocott methenamine silver (GMS)-stained slides from 205 bronchial specimens submitted for suspected opportunistic lung infection. FACs containing sporozoites were seen in 86 cases, all with positive GMS. FACs were absent in 119 cases with negative GMS. In patients previously treated for PCP, Pap showed FAC-like material which lacked sporozoites. GMS demonstrated clumped degenerated Pneumocystis carinii (PC) cysts. In 17 GMS-positive cases there were no FACs on Pap due to sampling error and low burden of organisms. Morphologic features of macrophages in these cases can suggest the presence of PC. When FACs are lacking, a special stain is required. When GMS is used, the control must contain PC to avoid a false negative GMS. The inconsistent uptake of GMS by PC is a pitfall which is distinct from sampling error and which to our knowledge has not been previously reported.

Detection of human cytomegalovirus infection in paraffin sections by Grocott’s methenamine silver stain for light and electron microscopy

Grocott's methenamine silver stain (GMS) has been used histologically for identifying Pneumocystis carinii and fungal infections in paraffin sections of surgical specimens. However, current studies indicate that aggregates of human cytomegalovirus (HCMV) in the cytoplasm of infected cells are also stained by GMS and can be detected by LM. Since precipitated silver particles are electron dense, the presence of silver labelled HCMV virions can be easily located and confirmed by TEM.GMS and light green stained paraffin sections positive for HCMV can be processed for TEM either by conventional procedures or as follows: Coverslips are removed by immersion in xylene. A few drops of 1% 0s04, freshly dissolved in xylene, are then pipetted onto each slide for 5 minutes. Prolonged osmication is to be avoided as it will cause reduction of the silver deposits. Slides are then rinsed in xylene and changed to 100% acetone. Substitution of acetone for xylene prior to resin infiltration will produce a better quality block for sectioning. The sections are embedded, polymerized and subsequently separated from slides as previously described. The light green staining is retained in the epoxy block, which facilitates tissue orientation and selection of areas for thin-sectioning.