Grocott's Methenamine Silver Stain Research Articles

When a Sore Throat is Not Your Average Sore Throat: A Case of Crohnʼs Epiglottitis

Crohn's disease (CD) is an autoimmune condition that affects the gastrointestinal tract, extending from the mouth to the anus. While CD is often seen in the small bowel and colon, oropharyngeal CD can be difficult to differentiate from other, more common oropharyngeal pathologies and can be refractory to treatment. We present a case of a 41 year old man with a history of CD who presented with 2 weeks of throat pain. The patient's CD was diagnosed 7 years prior and he had refractory symptoms despite treatment with infliximab and combination adalimumab with azathioprine. In the emergency room, the patient was found to be febrile to 39.3 with an exam remarkable for a muffled voice and erythematous oropharynx. He was started on acyclovir for suspected viral epiglottis. ENT performed a flexible laryngoscopy, which showed apthous ulcers on the lips, hard and soft palate and tonsils with epiglottic edema and pooling of secretions. He was started on broad-spectrum antibiotics. His symptoms improved slightly and he was discharged. However, four days later, the patient returned with worsening odynophagia and fevers. A repeat laryngoscopy showed extensive white ulcerative lesions along the epiglottis as well as the palate. Biopsies were obtained. He was restarted on broad-spectrum antibiotics, antifungals and steroids as well as an induction dose of ustekinumab given concern for involvement of his underlying CD. His symptoms improved significantly. Biopsy of the epiglottic area showed ulcerated mucosa with chronic inflammation, negative by Grocott methenamine silver stain, consistent with a diagnosis of Crohn's epiglottitis (Fig 1). CD commonly affects the small bowel and colon, but can be seen in the esophagus, stomach and oropharynx. Oral lesions are more common in patients with CD rather than ulcerative colitis and are more prevalent in younger patients. In addition, patients with more proximal CD and those with perianal disease are more likely to have oral manifestations. While oral CD occurs in roughly 20% of patients, Crohn's epiglottitis is incredibly rare. This case suggests that any patient with recurrent oral symptoms such as aphthous ulcers or persistent epiglottitis should undergo endoscopy to obtain a pathologic diagnosis. While oral infections are common in immunosuppressed patients, it is important to consider oral CD in patients with persistent symptoms and those resistant to common antibacterial and antiviral therapies.

Three-dimensional ultrastructural study of the anther of Silene latifolia infected with Microbotryum lychnidis-dioicae.

When Microbotryum lychnidis-dioicae infects a male Silene latifolia, M. lychnidis-dioicae smut spores develop in the pollen sac instead of pollen. In contrast, when M. lychnidis-dioicae infects a female S. latifolia, the female flowers become male-like, promoting stamen formation. However, it is unclear when and how M. lychnidis-dioicae invades the anther. It is important to investigate not only whether hyphae exist when the apical meristem tissue differentiates into flowers and anthers, but also whether hyphae exist when stamen filaments form. We used Grocott’s methenamine silver stain and lectin stain, which stain chitin in the fungal cell wall, to search for M. lychnidis-dioicae in flower tissues. A few M. lychnidis-dioicae hyphae were observed intercellularly in the center of the connective of vascular bundles at the early anther developmental stage. Subsequently, large numbers of deeply stained M. lychnidis-dioicae hyphae were observed intercellularly in the cells surrounding the pollen sac, as well as in the center of the pollen sac. Hyphae stained with lectin were observed intercellularly in all of the stamen filaments at flower development stages. Hyphae were observed in the peduncle connecting the flower and stem. It is thought that M. lychnidis-dioicae invaded the anther via the stamen filament over a long period. Additionally, in total, 163 sections of connective were obtained, and the cell structure of each anther was colored and subjected to three-dimensional reconstruction. The M. lychnidis-dioicae hyphae observed in the connective were mainly old hyphae with large vacuoles or dead hyphae (S1 Fig). These hyphae branched out, towards the pollen sac, while growing between the cells. We also observed that the host cells that collapsed near the hyphae had thick cell walls and teliospores. Cell wall collapse and cell degeneration were observed only around hyphae with thick cell walls.

Histopathology of fungal diseases of the lung.

Fungal pneumonias can be a diagnostic problem. However, their recognition is important as they can pose a significant health risk, especially in the immunocompromised host. While many of these infections are accompanied by necrotizing or non-necrotizing granulomas, some might be characterized by cellular interstitial pneumonia, intra-alveolar frothy material or only minimal inflammatory change. Much of the tissue reaction is dependent on the immune status of the patient and the type of fungal organism. While many of the fungi can be identified in tissue, especially if using histochemical stains such as Grocott's Methenamine Silver (GMS) stain and/or Periodic Acid Schiff (PAS) stain, in some cases, these stains are negative and the organisms can only be identified in cultures or using special techniques such as PCR or fungal serology. Some fungi can be accurately identified in tissue based on morphologic features; others require culture for exact classification. Knowledge about immune status, geographic region and social history of the patient are helpful in identifying the fungus and, therefore, detailed clinical and travel histories are important. In this manuscript we aim to describe the most common fungal infections that occur in the lung, their morphologic features, and differential diagnoses.

Aberrant staining with Grocott's methenamine silver: utility beyond fungal organisms

Historically, Grocott's methenamine silver (GMS) stain has been used in cytopathology to highlight Pneumocystis jiroveci and other fungal organisms. Several nonfungal organisms, however, can show distinct GMS staining patterns that are important to recognize. We prospectively and retrospectively identified nonfungal pathogenic organisms on GMS-stained liquid-based and cytospin preparations of respiratory cytologic specimens. The organisms included parasitic worms, viruses, and assorted bacteria. Nine cases were identified, including two cases each of Strongyloides stercoralis, Cytomegalovirus, Mycobacterium tuberculosis, Nocardia species, as well as one case of anthrax-like Bacillus cereus. The nonfungal organisms had silver deposition in varying locations including the internal organs and/or cuticle of Strongyloides stercoralis larvae, the intranuclear inclusions of Cytomegalovirus infected cells, the surfaces of partially acid-fast Nocardia species and acid-fast Mycobacterium tuberculosis, and the cell walls and central endospores of Bacillus cereus. In 3 of the 9 cases, organisms were not clinically suspected. It was the aberrant GMS staining that pointed to the diagnosis and led to the performance of the definitive stain, culture, or other test. GMS is a chromic acid, sodium bisulfate stain that precipitates silver ions in fungal polysaccharide walls, producing the characteristic black stain on light microscopy. It is helpful to recognize aberrant GMS staining to avoid misdiagnosis of fungal elements. GMS stains several nonfungal human pathogens and may be a particularly useful diagnostic aid when the infectious condition is not clinically suspected or the number of organisms is sparse and otherwise difficult to visualize by routine staining methods.

Rhinofacial Conidiobolomycosis in a 16-Year-Old Girl

Objectives : To present the case of a 16-year old girl with progressive facial disfigurement spanning 11 months due to Conidiobolomycosis.
 Methods: 
 Design: Case Report
 Setting: Tertiary Government Hospital
 Patient: One
 Results: A 16-year-old girl presented with a severe facial deformity of 11 months duration. The lesion started as a swelling in the right nasal vestibule, which later involved the entire nose, forehead, cheeks, upper and lower lip. A series of tissue biopsies revealed varied results -- chronic inflammation, chronic granulomatous inflammation with foreign body type giant cells, and eosinophilic granuloma—resulting in delayed provision of appropriate treatment. On the fourth biopsy using Grocott methenamine silver staining technique, septate fungal hyphae were identified. With a diagnosis of rhinofacial conidiobolomycosis, she was started on Itraconazole 100mg three times daily for eight months. Her facial swelling subsided gradually during the course of treatment and no systemic drug-related complications were observed.
 Conclusion: Rhinofacial conidiobolomycosis is a rare chronic localized fungal infection that usually affects midline facial structures in immunocompetent hosts. Early detection and diagnosis, and appropriate medication can give rapid resolution. To the best of our knowledge, this may be the first documented case of rhinofacial conidiobolomycosis in the Philippines.
 
 Keywords: Conidiobolomycosis; Conidiobolus; fungal infection; Itraconazole, Therapeutic use

Cross-reactivity in allergic fungal sinusitis. Case report

The presence of allergic mucin in allergic fungal sinusitis (AFS) is a manifestation that identifies it as a hypersensitivity process. AFS has a phenomenon of cross-reactivity to IgE-bound proteins having at least two shared epitopes. A 13-year-old male with nasal obstructive symptoms of three years of evolution. An obstructive mass was identified in the sinuses through physical examination and CT. In endoscopic surgery, the left nostril polyp was identified with the macroscopic appearance of allergic mucin; the polyp was resected. Final histopathological examination using periodic acid-Schiff and Grocott's methenamine silver staining indicated Aspergillus. Two weeks after surgery, percutaneous tests showed sensitization to Alternaria, Helminthosporium sativum, and Deramatophagoides farianae with negativity to Aspergillus fumigatus. The absence of significant titers of specific IgE antibodies to Aspergillus fumigatus was the evidence that the hypersensitivity response was triggered by a pathogen other than that isolated in histopathological study, which coupled with positive tests for other fungi may be explained by the cross-reactivity phenomenon in a phenomenon of likely hypersensitivity.

Chronic Invasive Pulmonary Aspergillosis in Two Cats with Diabetes Mellitus

Human patients with diabetes mellitus are at increased risk of fungal infections. Diabetes mellitus has also been implicated as a predisposing factor in the establishment of fungal lung infections in cats. Two diabetic cats of different origins presented with severe acute respiratory conditions that resulted in their death. At necropsy examination there was friable, black material in the main bronchi that obstructed the bronchial lumina. Microscopical examination of the lungs revealed the presence of pneumonia, calcium oxalate crystals and a large quantity of fungal hyphae and conidial heads. Fungal infection was confirmed with Grocott's methenamine silver stain. The results of the mycology analysis were compatible with Aspergillus section Nigri.

Aspergillus infection monitored by multimodal imaging in a rat model.

Although myriads of experimental approaches have been published in the field of fungal infection diagnostics, interestingly, in 21st century there is no satisfactory early noninvasive tool for Aspergillus diagnostics with good sensitivity and specificity. In this work, we for the first time described the fungal burden in rat lungs by multimodal imaging approach. The Aspergillus infection was monitored by positron emission tomography and light microscopy employing modified Grocott's methenamine silver staining and eosin counterstaining. Laser ablation inductively coupled plasma mass spectrometry imaging has revealed a dramatic iron increase in fungi-affected areas, which can be presumably attributed to microbial siderophores. Quantitative elemental data were inferred from matrix-matched standards prepared from rat lungs. The iron, silver, and gold MS images collected with variable laser foci revealed that particularly silver or gold can be used as excellent elements useful for sensitively tracking the Aspergillus infection. The limit of detection was determined for both (107) Ag and (197) Au as 0.03 μg/g (5 μm laser focus). The selective incorporation of (107) Ag and (197) Au into fungal cell bodies and low background noise from both elements were confirmed by energy dispersive X-ray scattering utilizing the submicron lateral resolving power of scanning electron microscopy. The low limits of detection and quantitation of both gold and silver make ICP-MS imaging monitoring a viable alternative to standard optical evaluation used in current clinical settings.

Fatal pyogranulomatous myocarditis in 10 Boxer puppies.

Over a period of 5 years, 10 pure-bred Boxer puppies, 9-16 weeks old, were presented with a history of sudden death and were diagnosed with pyogranulomatous myocarditis. The myocarditis was characterized by a mixed infiltrate composed predominantly of neutrophils and macrophages. In our retrospective study, original case records and archived materials were examined. All dogs were positive for Borrelia burgdorferi on immunohistochemistry (IHC). There was no evidence of infectious agents in formalin-fixed, paraffin-embedded (FFPE) heart tissue sections stained with hematoxylin and eosin, Ziehl-Neelsen, Gram, Grocott methenamine silver, Warthin-Starry, Von Kossa, and Steiner-Chapman stains. IHC for Chlamydia sp., Toxoplasma gondii, Neospora caninum, West Nile virus, and canine parvovirus also yielded a negative result in all dogs. Polymerase chain reaction testing for vector-borne pathogens on heart tissue from 9 of the dogs (1 frozen and 8 FFPE samples) yielded positive results for 1 dog with B. burgdorferi as well as Anaplasma phagocytophilum in another dog. Subsequently, 2 additional cases were found in a French Bulldog and a French Bulldog-Beagle mix that had identical morphology, test results, age, and seasonality to these 10 Boxer dogs. The similarities in the seasonality, signalment of the affected dogs, and the gross and microscopic lesions suggest a common etiology. Positive IHC and morphologic similarities to human Lyme carditis indicate that B. burgdorferi is likely the agent involved. An additional consideration for these cases is the possibility of a breed-specific autoimmune myocarditis or potential predisposition for cardiopathogenic agents in young Boxers.

Usefulness of lymphoid granulomatous inflammation culture obtained by endobronchial ultrasound-guided transbronchial needle aspiration in a fungal endemic area.

Background and Objectives:Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is the procedure of choice for the evaluation of mediastinal/hilar lymph node enlargements. Granulomatous inflammation of the mediastinal/hilar lymph nodes is often identified on routine histology. In addition, mediastinal lymphadenopathy may be present with undiagnosed infection. We sought to determine the usefulness of routine cultures and histology for infectious etiologies in a fungal endemic area when granulomatous inflammation is identified.Materials and Methods:We identified 56 of 210 patients with granulomatous inflammation on EBUS-TBNA biopsies from October 2012 through October 2014. An onsite cytologist evaluated all biopsies and an additional TBNA pass for microbiologic stains and cultures were obtained in those with granulomatous inflammation.Results:Of the 56 patients with granulomatous inflammation, 20 patients had caseating (necrotizing) granulomas while noncaseating (nonnecrotizing) granulomas were detected in 36 of the remainder patients. In patients with caseating granulomas, fungal elements were identified in 6 of 20 (30%) patients (histoplasma; N = 5, blastomyces; N = 1) on Grocott methenamine silver (GMS) stain. Lymph node cultures identified 3 of 20 (20%) patients as being positive for Mycobacterium tuberculosis (N = 1), Histoplasma capsulatum (N = 1), and Blastomyces dermatitidis (N = 1). Among patients with noncaseating granulomas, only 2 out of 36 (5%) were positive for fungal elements on GMS stain, identified as Histoplasma, although the lymph node cultures remained negative.Conclusion:The incidence of granulomatous inflammation of mediastinal lymph nodes was 26.6% in our series. Of these patients, noncaseating granulomas were more common (64% vs. 36%). Infectious organisms, fungal or acid-fast bacilli (AFB), on either staining or lymph node culture were rarely identified in noncaseating granulomas, 5% and none, respectively. Caseating granulomas were more commonly associated with positive lymph node fungal stain and culture, 35% and 15%, respectively. In a fungal endemic area, lymph node staining and culture can be considered in cases with caseating granulomatous inflammation, if known at the time of biopsy.

Topics

Fungal pneumonias can be a diagnostic problem. However, their recognition is important as they can pose a significant health risk, especially in the immunocompromised host. While many of these infections are accompanied by necrotizing or non-necrotizing granulomas, some might be characterized by cellular interstitial pneumonia, intra-alveolar frothy material or only minimal inflammatory change. Much of the tissue reaction is dependent on the immune status of the patient and the type of fungal organism. While many of the fungi can be identified in tissue, especially if using histochemical stains such as Grocott's Methenamine Silver (GMS) stain and/or Periodic Acid Schiff (PAS) stain, in some cases, these stains are negative and the organisms can only be identified in cultures or using special techniques such as PCR or fungal serology. Some fungi can be accurately identified in tissue based on morphologic features; others require culture for exact classification. Knowledge about immune status, geographic region and social history of the patient are helpful in identifying the fungus and, therefore, detailed clinical and travel histories are important. In this manuscript we aim to describe the most common fungal infections that occur in the lung, their morphologic features, and differential diagnoses.

A Case That Breaks the Mold: Gastric Aspergillosis as a Cause of Recurrent Bleeding

Invasive gastric aspergillosis is a rare cause of recurrent bleeding in immunocompromised patients. A 44-year-old man presented with severe sepsis from multi-lobar pneumonia requiring intubation, pressors, and dialysis. He was on IV methylprednisolone 40mg every 6 hours without PPI. On hospital day 9, he developed massive hematemesis. Following transfusion, esophagogastroduodenoscopy (EGD) revealed multiple ulcerations (largest 2 cm) in the gastric body along lesser and greater curves extending into the antrum. Several were oozing or had a visible vessel and were treated with epinephrine injection, gold probe cautery, and clipping. A PPI drip was started. Abdominal angiogram did not reveal extravasation of blood. However, over the next 5 days, he developed recurrent bleeding with 3 additional endoscopies with the same findings and interventions. After each EGD, hemostasis was achieved for 2-3 days but recurred, warranting repeat EGD. Due to lack of response to endoscopic therapy, he underwent total gastrectomy with Roux-en-Y esophagojejunostomy. Gastric ulcer biopsies showed fungal hyphae on Grocott's methenamine silver (GMS) stain. Immunostains were negative for Helicobacter pylori, CMV, and HSV. The gastrectomy specimen showed multiple superficial ulcerations throughout the stomach with pseudohyphal elements. Sputum cultures grew Aspergillus fumigatus and Aspergillus flavus. Therapy with liposomal amphotericin B and intravenous isavuconazole was begun and subsequently changed to oral voriconazole, with plans to continue for at least 3 months. He tested negative for causes of immune deficiency. Invasive aspergillosis typically develops in immunocompromised patients and carries a 50-60% mortality. Most commonly, invasive aspergillosis involves the lungs and rarely the gastrointestinal tract. The 6 reported cases of gastric aspergillosis presented as either ulcers or transmural necrosis. Most had previous chemotherapy or systemic steroid exposure. Four showed deep invasion transmurally or into the blood vessels; however, one case had pulmonary Aspergillus and superficial colonization of the gastric ulcers. Voriconazole is the recommended treatment for invasive aspergillosis and usually requires months to years of treatment. This individual worked in a nursery potting plants, which was likely his exposure. We believe he presented with pulmonary aspergillosis, became immunocompromised from the steroids, and this resulted in secondary infection of the stomach.

Adult Living-Donor Liver Transplantation for a Recipient With a High Preoperative 1,3-Beta-d-Glucan Level and Positive Test Result for Aspergillus Antigen

The patient was a 45-year-old man with underlying alcoholic liver cirrhosis. Two years prior, he was repeatedly hospitalized for liver failure symptoms and requested a living-donor liver transplantation (LDLT) because of end-stage cirrhosis. A pretransplantation blood test revealed a high 1,3-beta-d-glucan (BDG) value of 102.0 pg/mL (reference value <20.0 pg/mL) and a high blood Aspergillus antigen (AsAg) value of 1.6 cutoff index (COI; reference value <0.5 COI). Contrast-enhanced thoracoabdominal-pelvic computed tomography (CT) and cranial magnetic resonance imaging revealed no fungal infection. However, latent fungal infection could not be ruled out, hence preoperative antifungal agent treatment was administered. BDG and AsAg levels showed a decreasing trend after treatment initiation. However, normalization did not occur; the BDG and AsAg levels were 25.8 pg/mL and 1.0 COI, respectively. Although the possibility of latent fungal infection was judged low, we prophylactically administered antifungal agents after LDLT. The BDG level consistently increased at 35–39 pg/mL until postoperative day 5 but subsequently normalized. The AsAg level was higher than the limit of detection at 5.0 COI on postoperative day 3 but normalized to 0.2 COI on postoperative day 5 and did not subsequently increase. The postoperative course was uneventful despite bacterial pneumonia and the patient was discharged on postoperative day 35. A histopathologic examination (Grocott methenamine silver staining) and a fungal polymerase chain reaction assay were performed for the resected liver, but the results of both were negative. At 9 postoperative months, the patient was making ambulatory follow-up visits. Currently, the BDG and AsAg values remain normal and clinical progress is favorable. We found no reports of LDLT for a recipient with a high preoperative BDG level and positive test result for AsAg. Thus, we report on such a case with a discussion of the literature on the causes of high preoperative BDG and AsAg values.

Differences in candidal carriage and speciation among diabetic smokers, diabetic nonsmokers and controls: A study

Aim: The aim of the study was to evaluate the difference in carriage and species recognition of Candida in diabetic smokers, diabetic nonsmokers, and healthy controls. Materials and Methods: Nonstimulated whole saliva was used from 75 subjects. Imprint culture and cytosmears were sampled from the buccal mucosa. Isolation was performed on Sabouraud's dextrose agar media and various biochemical tests were performed for identification of Candida species. Cytosmears were stained by periodic acid Schiff (PAS) and Grocott's Methenamine silver (GMS) stains to evaluate the presence of candidal organisms. Results: Candidal isolation was more in diabetic smokers (72%). Candida albicans was the dominant species in all subjects (84.7%). GMS stain (74.66%) was more specific than PAS stain (29.33%) in fungal identification of cytosmears. Conclusion: The findings of the present study suggest that alterations in oral mucosa of diabetic smokers could provide increased attachment of Candida . Grocott's stained smears demonstrated Candida more readily than PAS stained smears and hence is suggested for a wider use.

A Case of the Hiccups in the Setting of Colonic Ulcers

Question: A 51-year-old seasonal farm worker with the human immunodeficiency virus (HIV; CD4 count, 122 cells/mm3) presented with persistent unexplained diarrhea, fevers, and hiccups. Although he had no pulmonary symptoms, the admission chest radiograph revealed bilateral miliary nodular opacities that were concerning for granulomatous processes including tuberculosis and fungal disease (Figure A). Sputum analysis and subsequently bronchoscopy with extensive brushing revealed no evidence of acid-fast bacilli. He was anemic with a hemoglobin of 8.4 g/dL and his albumin level was decreased at 2.8 g/dL. Blood, stool, and urine cultures to evaluate his recurrent fevers were also noncontributory; thus, more extensive imaging was obtained. Abdominal CT demonstrated bowel thickening of the right hemicolon and proximal transverse colon as well as adjacent inflammatory changes (Figure B). Subsequent colonoscopy revealed multiple, small ulcers (Figure C). Biopsy of the ulcer edges was performed and histology is presented in Figure D–G. What is the diagnosis? See the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. On pathologic examination (Figure D–G) spherules were seen on Grocott's methenamine silver stain consistent with the fungus Coccidioides immitis. Serum coccidioides titer via complement fixation was significantly elevated at 1:64. He was started on fluconazole and his symptoms promptly abated. Disseminated coccidioidomycosis rarely presents with gastrointestinal symptoms.1Adam R.D. Elliott S.P. Taljanovic M.S. The spectrum and presentation of disseminated coccidioidomycosis.Am J Med. 2009; 122: 770-777Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Diarrhea illness is even more unusual; those with gastrointestinal manifestations almost always present with peritonitis.2Smith G. Hoover S. Sobonya R. et al.Abdominal and pelvic coccidioidomycosis.Am J Med Sci. 2011; 341: 308-311Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar It is postulated that an immune response mediated by intrinsic CD-8 T cells, natural killer T cells, and pre-mast cells in the lamina propria protects the hollow luminal organs of the gastrointestinal tract from fungal pathogens. Given the nonspecific presentation, a high index of suspicion is critical to obtain the appropriate histologic stains and serologic tests. Given its rarity the sensitivity of stool studies for coccidioidomycosis is unknown, but is likely poor based on its performance in other fungal gastrointestinal infections in HIV-positive patients.3Cello J.P. Day L.W. Idiopathic AIDS enteropathy and treatment of gastrointestinal opportunistic pathogens.Gastroenterology. 2009; 136: 1952-1965Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar The patient’s history of HIV and vocation as a seasonal farm labor in California’s San Joaquin Valley prompted the appropriate evaluation.

Loop-mediated isothermal amplification method for diagnosing Pneumocystis pneumonia in HIV-uninfected immunocompromised patients with pulmonary infiltrates.

Loop-mediated isothermal amplification (LAMP) is becoming an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. We retrospectively evaluated 78 consecutive HIV-uninfected patients who underwent LAMP method for diagnosing Pneumocystis pneumonia (PCP). Diagnosis of PCP was made by the detection of Pneumocystis jirovecii (P. jirovecii) with positive LAMP or conventional staining (CS) (Grocott methenamine silver staining or Diff-Quick™) on the basis of compatible clinical symptoms and radiologic findings. Additionally, we reviewed HIV-uninfected immunocompromised patients who underwent subcontract PCR as a historical control. LAMP was positive in 10 (90.9%) of 11 positive-CS patients. Among 13 negative-CS patients with positive LAMP, 11 (84.6%) had PCP, and the remaining 2 were categorized as having P. jirovecii colonization. LDH levels in negative-CS PCP were higher than in positive-CS PCP (p = 0.026). (1 → 3)-β-D-glucan levels in negative-CS PCP were lower than in positive-CS PCP (p = 0.011). The interval from symptom onset to diagnosis as PCP in LAMP group (3.45 ± 1.77 days; n = 22) was shorter than in subcontract PCR group (6.90 ± 2.28 days; n = 10; p < 0.001). As for patients without PCP, duration of unnecessary PCP treatment in LAMP group (2; 2-3 days; n = 10) was shorter than in subcontract PCR group (7; 7-12.25 days; n = 6; p = 0.003). LAMP showed higher sensitivity (95.4%) and positive predictive value (91.3%) than subcontract PCR did. Pneumocystis LAMP method is a sensitive and cost-effective diagnostic method and is easy to administer in general hospitals. In-house LAMP method would realize early diagnosis of PCP, resulting in improving PCP prognosis and reducing unnecessary PCP-specific treatment.

Mucormycosis with diabetic and lupus nephropathy.

A 67-year-old woman with Stage 3 chronic kidney disease (CKD) due to systemic lupus erythematous (SLE). She was admitted to our hospital with a diagnosis of acute kidney injury. She had a past medical history of hypertension, acanthosis nigricans, obesity, obstructive sleep apnoea and type 2 diabetes mellitus with diabetic retinopathy. Two months prior to admission she had presented with a deterioration in renal function with proteinuria, which was interpreted as a flare of her SLE and she had received treatment with cyclophosphamide and i.v. 6-methylprednisolone. Five days after admission she presented with dyspnoea with bilateral pulmonary infiltrates. A diagnosis of heart failure was made and she underwent ultrafiltration with slight improvement. On the 10th day after admission she developed a right third cranial nerve palsy. A brain CT scan and lumbar puncture were normal except for the findings suggestive of chronic maxillary sinusitis. Fibrobronchoscopy showed necrosis of the nasal septum and infection due to Mucor genera was suspected. She underwent surgical debridement and initiated treatment with amphotericin B and caspofungin and was admitted to the ICU for respiratory support. The patient deteriorated despite treatment and died on her fourth day in the ICU. Rhizopus spp was identified in the oral cavity biopsies (Figure 1). Fig. 1. Necrosis of the oral cavity due to Rhizopus (green arrows). As the number of immunocompromised patients increase, the frequency of mucormycosis [1] is increasing. Mucormycosis, also known as zygomycosis, is an opportunistic fungal infection caused by organisms of the mucorales order. The most frequent species are Rhizopus, Mucor and Rhizomucor (Figure 2). Fig. 2. Rhizopus in oral cavity biopsy (green arrows). Haematoxylin and eosin stain. Risk factors [2] for these infections include diabetes mellitus (especially in patients with ketoacidosis), haematologic malignancies, haematopoietic stem cell transplantation, solid organ transplantation, deferoxamine treatment and iron overload (Figure 3). Fig. 3. Rhizopus in oral cavity biopsy (green arrow). Grocott's Methenamine Silver stain. Mucormycosis may present with rhino-orbital-cerebral (which is the most common), pulmonary, gastrointestinal, cutaneous, renal and disseminated involvement [3]. The diagnosis is based on identification of organisms in the tissue by histopathology with confirmation by culture. Treatment is based on a combination of surgical debridement with long-term amphotericin B therapy. Owing to the aggressive course of mucormycosis, even with treatment, many other treatments [4] have been considered such caspofungin (for rhino-orbital-cerebral disease), long-term posaconazol (as a step-down therapy) and deferasirox. The prognosis is poor in all the presentations with mortality rates as high as 87% with pulmonary involvement.

Histopathology of Pneumocystis carinii pneumonia in immunocompetent laboratory rats.

The occurrence of idiopathic pulmonary lesions in laboratory rats, characterized by lymphohistiocytic interstitial pneumonia with dense perivascular lymphoid cuffs, has been reported over the past decade. Although the term rat respiratory virus (RRV) was adopted to confer a putative viral etiology to the idiopathic pulmonary lesions, the etiology of this disease remains to be elucidated. Recently, inflammatory lesions have been observed in the lungs of immunocompetent laboratory rats similar to those previously described. Based on the latest evidence indicating that Pneumocystis carinii (P. carinii), and not putative RRV, causes infectious interstitial pneumonia in laboratory rats, the present study investigated whether the pulmonary lesions observed were caused by P. carinii infection. Male Sprague-Dawley rats, free of known pathogens, were introduced into a rat colony positive for RRV-type lesions. Routine histopathological examinations were performed on the rat lung tissues following exposure. The presence of Pneumocystis organisms was confirmed using Grocott’s methenamine silver (GMS) staining. At week 3 following introduction, a few small lymphoid aggregates were located adjacent to the edematous vascular sheath. By week 5, foci of dense perivascular lymphoid cuffing were observed. Multifocal lymphohistiocytic interstitial pneumonia and prominent lymphoid perivascular cuffs were observed between week 7 and 10. GMS staining confirmed the presence of Pneumocystis cysts. Thus, the results of the present study demonstrated that P. carinii caused lymphohistiocytic interstitial pneumonia in a group of laboratory rats. The observations strongly support the conclusion that P. carinii infection in immunocompetent laboratory rats causes the lung lesions that were previously attributed to RRV.

Utilização da técnica de imuno-histoquímica para confirmar casos de pitiose cutânea equina diagnosticados por meio de caracterização clínica e avaliação histopatológica

Avaliaram-se 12 feridas granulomatosas em membros torácicos e pélvicos de equinos da raça Pantaneira. Foi realizado diagnóstico sugestivo para pitiose cutânea de acordo com as características clínicas das feridas e avaliação histopatológica, com as colorações de hematoxilina-eosina (HE) e Prata Metenamina de Grocott (GMS). A confirmação do diagnóstico foi realizada por exame imuno-histoquímico, método streptavidina-biotina marcada (LSAB). O diagnóstico histopatológico foi de dermatite piogranulomatosa, focal extensa, acentuada, associada a "pseudo-hifas" características de Pythium insidiosum (pitiose cutânea), nos 12 animais desta pesquisa. O diagnóstico de pitiose foi confirmado em 100% dos casos pela imunomarcação positiva (LSAB) para Pythium insidiosum, caracterizada pela visualização de estruturas ramificadas e septadas, sendo observados 100% de paridade entre as técnicas. É possível concluir que a caracterização clínica das feridas granulomatosas com aspecto de pitiose em equinos associada ao resultado histopatológico sugestivo para pitiose equina constituem métodos de diagnóstico confiáveis, os quais podem ser confirmados pela técnica de imuno-histoquímica.

Equine Pulmonary Aspergillosis with Encephalitic, Myocardial, and Renal Dissemination

The cause of the death of a 16-month-old Brasileiro-de-Hipismo filly and a 3-year-old male Paint Horse with clinical manifestations of anemia and apathy from southern Brazil was investigated. These horses were maintained at the same stable; received hay as part of their diet and were submitted for routine necropsy evaluations. Significant gross findings included several nodules randomly distributed throughout the pulmonary lobes of both horses, and the kidneys, myocardium, and the frontal lobes of the cerebrum of the filly. Histopathological evaluation revealed pyogranulomatous bronchopneumonia in both horses; granulomatous interstitial nephritis, myocarditis, and encephalitis were observed in the filly. All lesions contained vasculitis and thrombosis associated with myriads of intralesional, branching, septate fungi consistent with Aspergillus spp.; intralesional fungi were more easily identified by the Grocott methenamine silver stain. Mycological culture of fresh pulmonary sections from both horses and the brain of the filly revealed pure growths of A. fumigatus. These findings confirmed the participation of A. fumigatus in the etiopathogenesis of the lesions observed in the lungs of both horses, and the cerebrum, myocardium and kidneys of the filly and might represent the first description of A. fumigatus-induced encephalitis in horses. Additionally, we believe that infection occurred during the ingestion of contaminated hay or by inhalation of spores within contaminated bedding that resulted in transient nasal mycosis, which progressed to pyogranulomatous bronchopneumonia in both horses with embolic encephalitic, myocardial, and renal dissemination of A. fumigatus occurring only in the filly.