Grocott-Gomori Methenamine Silver Stain Research Articles

Background: Gastrointestinal pythiosis, caused by Pythium insidiosum, is a severe and underdiagnosed disease in dogs, posing significant diagnostic and treatment challenges. Brazil ranks 2nd globally in reported pythiosis cases, with 29 cases occurring in dogs, which exhibited the highest fatality rate among reported cases in the country, with most showing gastrointestinal involvement. Understanding this condition’s epidemiology and diagnostic intricacies is crucial for improving management strategies and outcomes in affected animals. We aimed to elucidate the clinical presentation, diagnostic findings, and outcomes of a gastrointestinal pythiosis case in a young dog from Mossoró, Brazil. Case: A 1-year-and-1-month-old, 20 kg male mixed-breed dog presented with gastrointestinal symptoms (vomiting, diarrhea, hematochezia, and weight loss) following the rainy season in Mossoró, Brazil. The dog, which had access to a balcony and brick-paved yard, had no direct rain exposure but fell ill shortly after the rainy period. Initial veterinary examination revealed eosinophilia (3,432 eosinophils/mm³), suggesting bacterial or parasitic gastroenteritis. Treatment included deworming, enrofloxacin, and multivitamins, leading to initial improvement. However, symptoms recurred, and 5 months later, the dog exhibited worsened symptoms, including significant weight loss (from 20 kg to 13 kg) and increased eosinophilia (4,224 eosinophils/mm³), prompting further evaluation. Abdominal ultrasonography indicated thickened colon walls (0.99 cm), loss of wall stratification, and a suspected neoplasm (4.25 cm × 2.90 cm). Exploratory laparotomy revealed extensive intestinal adhesions and hypervascularization, leading to euthanasia due to poor prognosis. Necropsy revealed whitish necrotic areas in the colon and rectum with enlarged lymph nodes showing necrotic foci. Histopathological examination confirmed transmural pyogranulomatous inflammation with fibrous tissue proliferation and infiltrating macrophages, plasma cells, eosinophils, and neutrophils. Multinucleated giant cells surrounded caseous necrotic areas containing intralesional fungal hyphae (4–10 μm in diameter, irregular branching). Grocott-Gomori's methenamine silver (GMS) staining highlighted these hyphae, with strong immunostaining for P. insidiosum using immunohistochemistry. Discussion: This report describes a case of colitis, proctitis, and lymphadenitis in a young mixed-breed dog from Rio Grande do Norte's semi-arid region, caused by P. insidiosum infection confirmed via immunohistochemistry. Post mortem diagnosis, following exploratory laparotomy, revealed advanced intestinal involvement that precluded surgical resection, highlighting the critical need for early diagnosis to improve prognosis. A previous case in the same region involved anal mucocutaneous junction lesions treated with itraconazole and terbinafine. In this present case, clinical signs including vomiting, diarrhea, hematochezia, and weight loss initially suggested parasitic gastroenteritis. Histopathological analysis confirmed pyogranulomatous inflammation with eosinophilic infiltrates and necrotic areas indicative of P. insidiosum hyphae, visualized with GMS staining. Immunohistochemistry confirmed P. insidiosum involvement, which was essential for a definitive diagnosis. This case highlights the diagnostic complexities and severe outcomes of gastrointestinal pythiosis in dogs, emphasizing the need for early detection and precise management to improve treatment outcomes in affected animals. Keywords: fungal-like infection, gastrointestinal infection, histopathology, immunohistochemistry, oomycetes, pythiosis.

First confirmed case of equine pythiosis in Northern Veracruz, Mexico

Introduction: Dermatophytosis are very common fungal infections caused by the fungal species Microsporum, Epidermophyton or Trichophyton, which mostly affect the skin, the interdigital region, groin and scalp. Although they do not cause serious diseases, in patients with the human immunodeficiency virus the infection manifests itself and evolves exuberantly, usually with extensive and disseminated lesions. Objective: To review the literature on dermatophytosis in people living with human immunodeficiency virus and to present the experience in clinical care in a patient living with human immunodeficiency virus with extensive and disseminated dermatophytosis. Methods: A literature review on the topic was carried out in the PubMed/National Library of Medicine – USA databases, using the keywords dermatophytosis, or dermatophytosis associated with the words AIDS, human immunodeficiency virus or immunodeficiency, from 1988–2022. The clinical experience showed a patient living with human immunodeficiency virus developing AIDS and presenting with disseminated skin lesions. Samples of the lesion were collected by scraping, which were submitted to culture and there was growth of fungi of the Trichophyton sp genus. A biopsy of the lesion was also performed using the Grocott-Gomori's Methenamine Silver stain. Results: We found 1,014 articles, of which only 34 presented a direct correlation with our paper, and were used to discuss the main themes narrated in this article. We present clinical experience in the management of a patient with human immunodeficiency virus/AIDS and low adherence to antiretroviral treatment, showing extensive and disseminated erythematous-squamous lesions with a clinical diagnosis of tinea corporis, manifesting with a clinical picture usually not found in immunocompetent patients. The diagnosis was confirmed by laboratory tests with isolation of the Trichophyton sp fungus. The patient was treated with oral fluconazole, with complete remission of the clinical picture after two months. She was also thoroughly encouraged to use the prescribed antiretroviral medication correctly. Conclusion: Dermatophytosis in patients living with human immunodeficiency virus can present extensive and disseminated forms. The antifungal treatment is quite effective, with remission of the condition. Antiretroviral therapy is an important adjuvant for better recovery of the sickness.

Data on the genetic diversity of Pneumocystis jirovecii causing Pneumocystis pneumonia (PCP) among children are still limited, and there are no available data from the Indian subcontinent, particularly associations between genotypes and clinical characteristics. A total of 37 children (62 days-12 years [median 5.5 years]) were included in this study. Pneumocystis was diagnosed by microscopy using Grocott-Gomori methenamine silver stain in 12 cases and by nested PCR using mtLSUrRNA in 25 cases. Genotyping was performed using three different genes, mitochondrial large subunit ribosomal RNA (mtLSUrRNA), dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). mtLSUrRNA genotype 3 and novel mutations at the gene target DHFR (401 T > C) and DHPS 96/98 were frequently observed and clinically associated with severe PCP and treatment failure. Phylogenetic analyses revealed 13 unique sequence types (STs). Two STs (i) 3-DHFR 401 T > C-DHPS 96/98 – PJ1 and (ii) 3-DHFR 401 T > C-DHPS 96- PJ3 were significantly associated with treatment failure and high mortality among PCP-positive patients. In conclusion, the present study strongly suggests the emergence of virulent P. jirovecii strains or genetic polymorphisms, leading to treatment failure and high mortality. Our study is the first of its kind from the Indian subcontinent and has highlighted the genetic diversity of Pneumocystis jirovecii among children and their clinical outcomes. These findings emphasize the need to focus more on genotypes to better understand the epidemiology of Pneumocystis pneumonia.

46-Year-Old Man With Lower Back Pain

PP - ORAL PARACOCCIDIOIDOMYCOSIS: A CASE REPORT

Chronic skin ulcers in a patient returning from Mexico

Histoplasmosis is a world-wide distributed deep mycosis caused by Histoplasma capsulatum which has been endemic in many countries. We present a case involving an immunocompetent man evidencing the necessity of a multidisciplinary approach and rational requisition of exams. The disease has started as a pulmonary disease mimicking tuberculosis, although the exams have been negative. Immunodiffusion test indicate histoplasmosis, not confirmed by culture of sputum. After days the patient was forwarded by a private doctor for evaluation of oral lesions at our Department of Stomatology. An incisional biopsy revealed a nonspecific granulomatous inflammation and the Grocott-Gomori methenamine silver stain identified scarce oval structures that could represent fungal yeast. Sampling oral lesions with swab, it was observed the typical growth of H. capsulatum on culture. This case highlights the importance of doctor's integration diagnosing histoplasmosis, while a wide spectrum of clinical manifestations should be expected. Oral lesions may be the critical manifestation leading diagnosis.

Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations' (55th and 57th codon) association with prior sulfa prophylaxis failure has been reported from both developed and developing countries. We conducted a prospective study to determine the prevalence of P. jirovecii DHPS mutations from 2006 to 2009 on P. jirovecii isolates obtained from HIV-infected patients with a clinical diagnosis of Pneumocystis carinii pneumonia (PCP) admitted to our tertiary care reference health center in New Delhi, India. Detection of P. jirovecii cysts was performed by direct fluorescent antibody (DFA) staining and by Grocott's-Gomori methenamine silver staining (GMS). DNA detection was performed by polymerase chain reaction (PCR) using primers for the major surface glycoprotein (MSG) gene. P. jirovecii DHPS gene was amplified by nested PCR protocol and sequenced for detecting mutations at the 55th and 57th codons. Out of 147 HIV-positive patients with suspected Pneumocystis pneumonia (PCP), 16 (10.8%) PCP positive cases were detected. Of 16 cases, nine (56.2%) were positive by DFA staining, four (25%) were positive by Grocott's-Gomori methenamine silver staining, and all 16 were positive by MSG PCR. DHPS mutations at the 55th and 57th codons were observed in 6.2% of HIV patients studied, which was relatively low compared to reports from developed nations. Prevalence of Pneumocystis jirovecii DHPS mutations associated with cotrimoxazole treatment failure may be low in the Indian subpopulation of HIV-positive patients and warrants larger studies to elucidate the true picture of Pneumocystis jirovecii sulfa drug resistance in India.

Cytological, histopathological and immunohistochemical examinations were carried out on a presumed 10-year-old Japanese cat showing vomiting and emaciation. On cytologic examination of the mass of the upper abdominal cavity, many yeast-like organisms were detected in the macrophages. At necropsy, the upper part of colon was markedly dilated with a thickened wall. The lung did not show significant changes. Histologically, severe necrotic and granulomatous lesions were observed in the colon. In the colonic lesion, the cytoplasm of the macrophages contained yeast-like organisms with irregularly shaped dots, and the cell walls of these organisms were stained black by Grocott-Gomori methenamine-silver stain. Immunohistochemically, they were found to be positive for anti-histoplasma yeast antibody. This is the first report of feline histoplasmosis in Japan.

A 36-Year-Old Woman With a Unilateral Breast Mass

We examined four staining methods on replicate smears of 313 respiratory specimens submitted for Pneumocystis jiroveci examination. The sensitivity and specificity of Calcofluor white stain (CW) were 73.8 and 99.6%, respectively. The sensitivity and specificity of Grocott-Gomori methenamine silver stain (GMS) were 79.4 and 99.2%, respectively. The sensitivity and specificity of Diff-Quik stain were 49.2 and 99.6%, respectively. The sensitivity and specificity of Merifluor Pneumocystis stain were 90.8 and 81.9%, respectively. Only CW and GMS had positive and negative predictive values of >90%.

Pulmonary zygomycosis rarely occurs without pre-existing immunocompromised disease. A 72-year-old male was found to have a nodular shadow (3 cm x 4 cm) in the right S8 and S9 on a chest X-ray. Right lower lobectomy was performed and histological examination of the resected material demonstrated pulmonary zygomycosis. Hyphae stained positively not only with Grocott-Gomori methenamine silver staining, but also with an anti-Rhizopus oryzae polyclonal antibody.

The diagnosis of paracoccidioidomycosis was made in the case of a hospitalized patient in New Orleans. The biopsy showed multiple budding and variations in fungus cell size. In the hematoxylin-eosin sections, the budding was not obvious and the first suggestion offered was that it was North American blastomycosis. The Grocott-Gomori methenamine silver stain demonstrated the multiple budding and variation in cell size to better advantage. Cultures revealed<i>Paracoccidioides brasiliensis</i>, skin tests were positive to paracoccidiodin, and results of serologic tests were positive. The patient had worked in South America for a number of years, though he was a native of the United States.