Grocott Methenamine Research Articles

Fungi and bacteria occupy distinct spatial niches within carious dentin.

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal β-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.

Metagenomic Next-Generation Sequencing for Accurate Diagnosis of Pneumocystis jirovecii Pneumonia: A Comparative Study with Traditional Methods.

Metagenomic next-generation sequencing (mNGS) is a high-throughput sequencing technique that identifies a wide array of pathogens directly from clinical specimens. This study evaluates the diagnostic value of mNGS in Pneumocystis jirovecii pneumonia (PJP) and compares its efficacy with traditional detection methods, including Grocott's Methenamine Silver (GMS) staining, serum (1-3)-β-D-Glucan (BDG) testing, and Lactate Dehydrogenase (LDH) testing. Seventy-eight patients hospitalized between January 2022 and March 2023 with suspected pulmonary infections were included. Patients were eligible for mNGS if they exhibited symptoms such as fever, cough, dyspnea, or progressive hypoxemia, and met specific clinical criteria for PJP. Specimens obtained included bronchoalveolar lavage fluid, sputum, and peripheral blood. Positive rates and pathogen distributions detected by mNGS and traditional methods were compared. In the PJP group, 25%, 37.5%, and 9.38% of patients had solid organ tumors, corticosteroid use, and skin diseases, respectively, significantly higher than in the non-PJP group. The sensitivity and specificity of mNGS were both 100%, significantly higher than those of serum BDG (sensitivity 50%, specificity 81.8%) and LDH (sensitivity 9.3%, specificity 91.3%). Significant differences in microbial composition between the PJP and Non-PJP groups were observed. mNGS detected multiple mixed pathogens in 96.88% of PJP cases, with 68.75% exhibiting mixed bacterial and viral infections. Notably, 71% of patients improved following antibacterial treatment based on mNGS results. mNGS technology shows superior sensitivity and specificity in diagnosing PJP and guides precise treatment for complex pulmonary infections.

Silica and barium: Comparison of microscopic appearance and characterization of effects of silica on cytomorphology.

Silica from plastic red top sample collection tubes and barium cause recognized artifacts in slide preparations for microscopic examination. The objectives of this study were to evaluate and directly compare the microscopic appearance of silica and barium particles and various slide preparation techniques (e.g., use of coverslips, oil immersion, and different stains). A secondary objective of this study was to evaluate the effects of silica particles on cellular morphology after mechanical trauma with cytocentrifugation. Fluid samples (deionized water, pleural effusion, peritoneal effusion, cerebrospinal fluid, and urine) were collected and evaluated in silica- and non-silica-containing tubes. Barium was added to silica and non-silica samples. Direct and cytocentrifuge preparations were compared to evaluate the effect of silica particles on cellular morphology. Preparations were stained with Wright-Giemsa, rhodizonic acid disodium salt, Alizarin Red, Grocott's methenamine silver, and Prussian blue. Silica and barium particles were identifiable via light microscopy with and without polarized light, although silica particles diminished with immersion oil. Barium particles retained their structure and diminished less under oil. Cytoseal mounting medium for coverslip placement resulted in diminished refractility of silica and some barium particles. Silica particles with mechanical interaction during cytocentrifugation resulted in disrupted cellular morphology with many lysed cells. Silica and barium particles were negative for all special stains tested. Silica from plastic red top tubes adversely affects cell morphology in cytocentrifuge preparations, potentially affecting manual differential cell counts and compromising diagnostic interpretation. Samples intended for microscopic evaluation should not be collected in silica-containing tubes.

Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

ORAL INVOLVEMENT IN DISSEMINATED HISTOPLASMOSIS - A CASE REPORT

Histoplasmosis is a systemic mycosis. Oral involvement is very rare, usually present in cases of disseminated disease. A 71-year-old man complained of a painful mouth sore, with a 2-month duration. The patient reported a history of heavy tobacco consumption and alcoholism. Clinical examination revealed a large ulceration extending from the hard palate to the left alveolar ridge. Computed tomography revealed involvement of the alveolar bone, showing irregularity of the left posterior alveolar ridge. The clinical diagnosis was oral squamous cell carcinoma, and an incisional biopsy was performed. Histopathological examination showed inflammatory cells in the lamina propria and numerous, small round-shaped yeast-like bodies in the interior of vacuolated macrophages. Periodic Acid-Schiff and Grocott's methenamine silver stains disclosed positive results for round bodies, confirming the diagnosis of oral histoplamosis. The patient was hospitalized, and in a short period of time, he contracted disease-related systemic complications and died. Histoplasmosis is a systemic mycosis. Oral involvement is very rare, usually present in cases of disseminated disease. A 71-year-old man complained of a painful mouth sore, with a 2-month duration. The patient reported a history of heavy tobacco consumption and alcoholism. Clinical examination revealed a large ulceration extending from the hard palate to the left alveolar ridge. Computed tomography revealed involvement of the alveolar bone, showing irregularity of the left posterior alveolar ridge. The clinical diagnosis was oral squamous cell carcinoma, and an incisional biopsy was performed. Histopathological examination showed inflammatory cells in the lamina propria and numerous, small round-shaped yeast-like bodies in the interior of vacuolated macrophages. Periodic Acid-Schiff and Grocott's methenamine silver stains disclosed positive results for round bodies, confirming the diagnosis of oral histoplamosis. The patient was hospitalized, and in a short period of time, he contracted disease-related systemic complications and died.

Standardization of Grocott's methenamine (hexamine) silver method for glycogen demonstration in liver tissue.

Demonstration of glycogen can be done in different lesions and is considered diagnostically significant, mainly in some tumors. Glycogen staining is affected by the type of fixative, the temperature of fixation, and the staining technique.Grocott's methenamine (hexamine) silver technique quality was assessed after four different types of fixatives at two different temperatures [Bouin's solution, 10% neutral buffered formalin (NBF), 80% alcohol, and Rossman's solution at room temperature (RT) and 4°C, for 24h]. These variables were studied to optimize this technique for glycogen demonstration. Archived paraffin blocks were used in this study. They were prepared from one rabbit's liver, and 32 paraffin sections were prepared and stained with Grocott's methenamine (hexamine) silver technique. Eightypercent alcohol provided the highest staining quality scores at both RT and 4°C in comparison with the other fixatives. We concluded that 80% alcohol at 4°C seems to be the fixative of choice for glycogen with the Grocott's methenamine (hexamine) silver technique at the level of this study.

Sarcoidosis as an unusual cause of unexplained pancytopenia.

Sarcoidosis is a systemic inflammatory disease of unknown etiology characterized by noncaseating granulomata, most commonly in lung, skin, and lymph nodes. However, sarcoidosis can affect any tissue, including the bone marrow, although this finding is very rare (0.3%–2.2% of bone marrow biopsies) [1]. A 35-year-old male presented with hematochezia and hemoptysis. He was consequently found to have pancytopenia with diffuse lymphadenopathy and marked splenomegaly. The patient's complete blood count (CBC) was notable for severe panleukopenia with total white blood cell count of 1.45 × 10^3, normocytic, normochromic anemia with hemoglobin of 9.5 g/dl, and thrombocytopenia to 72 × 10^3. Blood cultures were negative for pathogenic organisms. Bone marrow biopsy was performed. Histological findings revealed noncaseating granulomata with moderate myelofibrosis involving approximately a third of the bone marrow (Figure 1A). Special stains for reticulin and trichrome showed moderate myelofibrosis (MF-2) (Figure 1B–C). CD68 highlights dense infiltration of histiocytes into the bone marrow and granulomata (Figure 1D). CD3, CD20, and CD56 highlight scattered infiltrating T- and B-lymphocytes, and natural killer (NK)-cells. Grocott's methenamine silver (GMS) and acid-fast bacteria (AFB)/Fite stains were negative for microorganisms. Next generation sequencing was performed and was negative for mutations in JAK2, MPL, and CALR. After extensive clinical history review, we discovered an extant diagnosis of sarcoidosis for which the patient was unable to access treatment. Based on this prior clinical history and our histological findings, the diagnosis of sarcoidosis involving bone marrow was established [2]. Granulomas are a nonspecific finding often seen in infections, particularly tuberculosis and fungal. In this case, all infectious work up—including GMS and AFB stains—was negative. Sarcoidosis classically involves lungs, skin, and lymph nodes. Involvement of the bone marrow is a rare finding, estimated to be present in only 10%–17% of all sarcoidosis cases [3]. This case highlights the progression of untreated sarcoidosis, ultimately leading to infiltration of the bone marrow. The resulting suppression of hematopoiesis is the likely cause of the patient's pancytopenia [4]. Following identification of sarcoidosis involvement of bone marrow, the patient was treated with high dose methylprednisolone. Following two weeks of treatment, CBC numbers improved significantly with white blood cells of 7.75 × 103, hemoglobin of 10.1 g/dl, and platelets of 145 × 103. Caitlin Raymond wrote the manuscript, assisted in taking original images, and produced the figure. Sir Kavuri assisted in taking original images, and provided valuable contributions on the manuscript. Faisal Rawas produced the slides from which images were taken. Jayati Mallick and Juan Olano provided valuable contributions to the manuscript. Kirill Lyapichev assisted in taking original images, and provided valuable comments on the manuscript. We would like to thank Dingge Li, Liesel Dell'osso, Donna Fuller, and Juan D. Garcia for technical professional support. There were no sources of funding for this manuscript. The authors have no conflict of interest to disclose. The information presented in this manuscript is deidentified, and there is minimal risk to the patient's privacy or confidentiality. IRB approval was not required by our institution for preparation of this manuscript. Patient consent was not obtained. No material from other sources is included in this manuscript. Data from this manuscript will be shared upon request

Histological, immunohistochemical and ultrastructural features of polyglucosan bodies in uterine smooth muscle of pet rabbits (Oryctolaguscuniculus)

We document the frequency and morphological and immunohistochemical features of inclusion bodies in uterine smooth muscle cells in 56 (76%) of 74 investigated pet rabbits (Oryctolagus cuniculus). Inclusion bodies began to appear at the age of 2 years and their frequency increased with age (P = 0.047, r = 0.33). They ranged from 5 to 20 μm in diameter, were slightly basophilic to amphophilic with well-delimited oval bodies in haematoxylin and eosin-stained tissue sections and formed in the cytoplasm of the uterine smooth muscle cells with displacement of the cell nuclei. The inclusion bodies were positive with periodic acid–Schiff, Best's carmine, Lugol's iodine and Grocott's methenamine silver methods. They were immunoreactive to a monoclonal antibody raised against human polyglucosan and negative with monoclonal antibodies for several intermediate filament proteins. Electron microscopy revealed that they were non-membranous structures composed of electron-dense amorphous material. The morphological, histochemical, immunohistochemical and ultrastructural features of the inclusion bodies in the rabbi uteri were similar to those of human polyglucosan bodies (PGBs). PGBs appear to occur at a high frequency in the uterus of rabbits, which are known to be susceptible to uterine diseases.

S2011 Disseminated Histoplasmosis Involving the Whole Colon: It's Ulcers All the Way Down

Introduction: Histoplasma capsulatum is a dimorphic fungus endemic to river valleys of Eastern and Central U.S. that is spread via inhalation of spores from soil contaminated by bird or bat droppings. Disseminated Histoplasmosis (DH) spreads through the reticuloendothelial system via parasitized macrophages and can cause severe infection in immunocompromised patients with AIDS, hematologic malignancies, transplant recipients, and those receiving tumor necrosis factor (TNF)-alpha inhibitors or chronic corticosteroids. Gastrointestinal involvement in DH is frequently seen, but it is not uncommon for patients to be asymptomatic. We present a case of DH with pan-colonic ulcerations in a renal transplant patient. Case Description/Methods: A 83-year-old male with deceased donor renal transplantation on immunosuppression therapies presented with profound weakness. He was restarted on hemodialysis one month prior for declining renal allograft function which had been stable for the past 2 decades. Initial workup showed pneumonia on imaging, elevated procalcitonin, alkaline phosphatase, and pancytopenia. He developed hematochezia with worsening anemia which prompted colonoscopy that revealed deep and innumerable ulcers measuring greater than one cm in the whole colon. One large ulcer with a visible vessel near the hepatic flexure was treated with epinephrine, electrocautery, and endoclip for hemostasis. Ulcer biopsy revealed granulomatous inflammation with fungal yeast forms morphologically compatible with Histoplasma by Grocott's Methenamine Silver (GMS) staining. Bone marrow pathology was suggestive of fungal infiltration, but marrow and blood cultures were negative. Family revealed he lives in an old mold infested home and has history of visiting caves. Treatment was initiated with liposomal Amphotericin B then transitioned to oral Itraconazole. He has continued to make a strong recovery and remains on dialysis (Figure). Discussion: For healthy individuals, exposures to Histoplasma generally occur as asymptomatic to self-limited pulmonary infection. Disseminated Histoplasmosis is often undiagnosed and carries high mortality if untreated. These patients usually had past exposures with dormant inoculation that is reactivated after immunosuppression. GI involvement is common but can be non-symptomatic or with only vague abdominal complaints from colonic fungal invasion. Suspicion of DH should be met with a low threshold for endoscopic evaluation and requires tissue biopsy for diagnosis.Figure 1.: (A) Large ulcer in the cecum which was biopsied. (B) Numerous ulcers seen diffusely throughout the colon. (C, D) H&E and Grocott’s Methenamine Silver (GMS) stain of colonic mucosa with patchy ulceration and granulomatous inflammation including fungal yeast forms, morphologically compatible with Histoplasma.

Host factors and characteristics of hospitalized patients with pneumocystis jirovecii pneumonia

Objectives: To describe the underlying diseases, microbiologic examination and severity of hospitalized patients with Pneumocystis jirovecii pneumonia (PJP) in a tertiary Chinese hospital. Methods: We conducted a retrospective analysis of 485 identified PJP patients who were admitted to our hospital between January 2013 and December 2021. Results: Among the 485 enrolled PJP cases, there were 237 males and 248 females, aging (53.3±16.2) years (range from 14 y to 88 y). They were divided into 8 subgroups with variable underlying diseases. There were 209 cases with connective tissue diseases(CTD), 27 cases with non-hematologic malignancies, 38 cases with hematologic malignancies, 81 cases with kidney diseases, 33 cases with idiopathic interstitial pneumonia(IIP), 30 cases infected with human immunodeficiency virus (HIV), and 42 cases with miscellaneous underlying diseases. In the CTD group, there was more females than males, while male patients were predominant in both the malignant and the HIV groups. The Pneumocystis was identified in 44.95%(218/485) sputum samples and 92.01%(265/288) bronchoscopic samples. Pneumocystis asci were observed at direct microscopic examination with Grocott's methenamine silver stain in 4.95%(24/485)sputum samples and 9.72%(28/288)bronchoscopic samples. Pneumocystis DNA fragments were identified by PCR analysis in 43.09%(209/485)sputum samples and 90.63%(261/288)bronchoscopic samples. Among the 8 groups, cytomegaviremia and respiratory failure were most common in the HIV-infected PJP group, but the rates of mechanic ventilation, intensive care unit (ICU) admission and death were the lowest. There were less PJP patients in the IIP group (IIP-PJP) who received mechanic ventilation and admitted to ICU than the other groups except HIV-infected PJP group. However, the mortality rate was highest for the IIP-PJP group. Conclusions: CTD was the most common predisposed underlying disease for our enrolled PJP cases. Cytomegaviremia and respiratory failure were common in HIV-infected PJP patients, but the prognosis of HIV-PJP was slightly better than the others. The disease was more severe, rapidly progressive and fatal in the IIP-PJP group.

A Rare Presentation of Sarcoidosis: Dysphagia, Pancytopenia, and Acute Renal Failure

Sarcoidosis is a multisystem inflammatory chronic disorder that can virtually affect any organ system in the body. Most commonly affected organs are the intrathoracic structures with 90% of the reported cases involving the lungs. Esophageal involvement in sarcoidosis is extremely rare. Involvement of the esophagus and kidney along with hematological involvement is extremely uncommon in the same patient. Here, we present a case of a 58-year-old gentleman with a similar rare presentation. The patient presented with shortness of breath, productive cough, fatigue, and difficulty in swallowing, along with a weight loss of 20-30 pounds over three months. Laboratory workup was significant for leukopenia (2900 K/UL), serum creatinine level of 2.7 mg/dL (baseline: 1.2-1.7), and raised angiotensin-converting enzyme level at 187 nmol/ml/min. Chest X-ray showed bilateral widespread fine reticulonodular opacities, chest CT showed extensive bilateral reticulonodular opacities throughout the lung parenchyma, and fine-needle aspiration cytology of the right lung showed noncaseating granulomas. No fungal or acid-fast organisms were identified, and no evidence of malignancy was seen. Special stains for fungal (Grocott's methenamine silver and periodic acid-Schiff) and acid-fast organisms (acid-fast bacilli (AFB) and fluorescent AFB) were negative. Esophagogastroduodenoscopy (EGD) with gastric biopsy showed acute and chronic inflammation and no intestinal metaplasia, dysplasia, or malignancy was identified. Bronchoalveolar lavage was done, which showed macrophages (74%), neutrophils (6%), eosinophils (3%), and lymphocytes (17%), and was negative for malignant cells. QuantiFERON and AFB sputum/Mycobacterium tuberculosis polymerase chain reaction were negative. The patient was initially started on intravenous fluids and calcitonin, which significantly improved renal function and the calcium status of the body. Then prednisone 40 mg daily was started, which improved swallowing and breathing. After a week, prednisone was changed to 20 mg daily and was continued at the time of discharge.

Biliary candidiasis mimicking IgG4‐related disease in a patient with normal immunity

AbstractPurposeTo report a rare case of biliary candidiasis in a host with normal immunity.MethodsLaboratory tests indicated an elevated G level of 1000 pg/ml. Microscopic examination of pathological sections demonstrated multiple, narrow base, budding yeast cells following Grocott's methenamine silver staining and periodic acid‐Schiff staining. Subsequent sequence analysis supported the diagnosis of biliary candidiasis.ResultsA 31‐year‐old previously healthy man developed obstructive jaundice under no obvious inducement. He experienced cholecystectomy and biliary drainage. Surgical specimens revealed granulomatous chronic inflammation in the gallbladder, liver, bile ducts and mesenteric lymph nodes. He was misdiagnosed with tuberculosis and received antituberculosis treatment. However, his condition did not improve. He was finally diagnosed with biliary candidiasis and administered the antifungal treatment with fluconazole. His symptoms improved after such treatment.ConclusionsWe reported a case of biliary candidiasis that mimicked IgG4‐RD in a host with normal immunity. Our findings highlighted the need for pathological diagnosis in patients with symptoms resembling IgG4‐RD.

Aerossacolitis and Pneumonia in an Indian Peafowl Caused by Lactobacillus agilis

Background: The peafowl is an ornamental bird that has the habit of eating directly from the earthy soil, which makes this bird more susceptible to endoparasites. One important endoparasite is Eucoleus contortus, which leads to inflammatory processes that alter the local microbiota, potentializing disease. By the other way, a member of the bird’s microbiota there is the genus Lactobacillus, but when occurs some imbalance, these bacteria can overgrowth and even cause some infection. This report describes the pathological and microbiological findings of chronic necrotizing pneumonia and aerossacolitis caused by Lactobacillus agilis in a peafowl, associated with parasitism by E. contortus. Case: A peafowl (Pavo cristatus), adult, male, who lived on a farm with contact with other species of animal, was submitted to post-mortem examination due to sudden death. This animal lived in an extensive system on the property and was the only one of its species. During the gross evaluation, the air sacs were filled with solid yellowish crumbly material. The same material was observed forming well-defined nodules that occupied > 50% of the lung parenchyma. Histological analysis showed multiple parabronchi dilated and filled with caseous necrosis, characterized by abundant cellular debris and fibrin deposition. These areas were surrounded by the proliferation of fibrous connective tissue and inflammatory infiltrate of macrophages, giant cells, lymphocytes, and plasma cells. The air sacs parenchyma showed fibrin deposition and mixed inflammatory infiltrate. Multiple gram-positive bacilli were observed within the caseous foci in Gram-stained slides. In the crop and esophageal mucosa, cross-sections of filiform nematodes morphologically compatible with E. contortus were associated with chronic inflammatory infiltrate and epidermal hyperkeratosis. A lung section was submitted to Gram-Brown-Hopps and Ziehl-Neelsen (ZN) stains for bacterial investigation, and Grocott's methenamine silver (GMS) stain for fungal investigation. Short gram-positive bacilli bacteria are observed within the caseous foci in Gram-stained slides. By the other way, no agents were identified on the ZN and GMS stains. Following the analysis, lung fragments were cultivated at aerobic and microaerophilic conditions on sheep blood agar and McConkey agar. All the microbial cultures were incubated at 37°C to 48 h. Pure culture, in microaerophilic condition, of Gram-positive bacilli, was observed. The isolated bacterium was identified by MALDI-TOF MS as L. agilis.Discussion: Although uncommon, L. agilis was the single bacterium identified and therefore, associated as a primary cause of necrotic pneumonia and aerossacolitis in the studied peafowl. The presence of E. contortus could induce the aspiration of regurgitated of little amount of material from the gastrointestinal tract with sufficient bacterial load to initiate an infection, but not enough to smother the animal. The gradual aspiration can induce a chronic inflammatory condition. Infections by bacteria from the host microbiota have been observed in animals and humans with immunodeficiency. In summary, both the parasitosis and the inflammation could be resulted by the parasite and the aspiration of gastric product, which probably interfered in the immune response and allowed the overgrowth of L. agilis. In the current case report, based on macroscopic, microscopic and bacteriological results, we have provided insights to understand how the parasitosis made possible a pneumonia from a bacterium from the host's microbiota. Finally, to the best of our knowledge, this is the first report of L. agilis as causal agent of fatal necrotic pneumonia and aerossacolitis in peafowl.Keywords: respiratory infection, ornamental birds, secondary infection, parasitosis.

Toxicity studies of Aspergillus fumigatus administered by inhalation to B6C3F1/N mice (revised).

Aspergillus fumigatus is a thermotolerant, soil-borne fungal species that is ubiquitous in the environment. Mold was nominated to the National Toxicology Program (NTP) by a private individual due to suspected adverse health effects associated with personal exposure in indoor and occupational settings. A. fumigatus is of particular concern in the biowaste industry as the species can contaminate self-heating compost piles. Because of this potential for personal and occupational exposure and the lack of available toxicity data, toxicity studies were conducted in which male and female B6C3F1/N mice were exposed to A. fumigatus conidia (spores) two times a week for 3 months. All in-life procedures, including inhalation exposure, test article preparation, and hematology analysis, were completed by the National Institute for Occupational Safety and Health (NIOSH, Morgantown, WV). Battelle (Columbus, OH) conducted terminal necropsies, measured terminal body and organ weights, and evaluated gross lesions on-site at NIOSH. Tissue processing and histopathology were completed at Battelle. Grocott's methenamine silver (GMS) staining was performed at NIOSH. Genetic toxicology studies on mouse peripheral blood erythrocytes were conducted by Integrated Laboratory Systems, LLC (Research Triangle Park, NC). (Abstract Abridged).

CUTANEOUS AND PULMONARY BLASTOMYCOSIS INFECTION: A CASE REPORT

<h3>Introduction</h3> Blastomycosis is a chronic granulomatous disease caused by <i>Blastomyces dermatitidis</i>. More frequently, it presents as a respiratory disease. However, extrapulmonary disease occurs in 25% to 30% of patients after hematogenous dissemination from the lungs. The most common site is the skin; bone, the genitourinary system, and the central nervous system can also be affected. <h3>Case Report</h3> A 34-year-old female patient from Oaxaca, México, presented with an asymptomatic ulcer, verrucous and eroded plaques, and scab areas of unknown evolution on the low and medium third of the face and the nose. The histopathologic staining with hematoxylin and eosin revealed a squamous epithelium with a pseudoepitheliomatous hyperplasia and granulomatous inflammation, containing numerous Langhans multinucleated giant cells and spherical microorganisms of approximately 12 µm characterized by a double-contoured yeast and thick birefringent wall. Periodic acid-Schiff and Grocott's methenamine silver special staining showed positive results for fungal organisms. The thoracic imaging revealed lung involvement. The histopathologic final diagnosis was blastomycosis. <h3>Discussion</h3> In Mexico from 1898 to 2017, only 6 cases were reported. Blastomycosis infections can clinically be easily mistaken for malignant neoplasms or other cutaneous infections more prevalent in Mexico, such as leishmaniasis, coccidioidomycosis, leprosy, or adiaspiromycosis. <h3>Conclusions</h3> The treatment of choice for all forms of the disease includes itraconazole 600 mg, oral 3 days, followed by 200 to 400 mg every day for 6 to 12 months. Spontaneous remissions have been reported in patients with a mild form of blastomycosis. Our patient had 6 months of follow-up with remission of the lesion

Pneumocystis jirovecii and microsporidia: An unusual coinfection in HIV patients?

Pneumocystis jirovecii and microsporidia species are recognized as opportunistic infectious pathogens in AIDS patients. Coinfection of both in one patient has been rarely reported. The aim of the present study was to investigate the coinfection of P. jirovecii and microsporidia in different tissues from AIDS deceased patients. Post mortem histological finding of P. jirovecii and microsporidia was demonstrated by means of the Grocott's methenamine silver and Brown Brenn staining, respectively. Molecular technique was used for identification and characterization of both fungi. Out of the 514 autopsied cases P. jirovecii and microsporidia species were identified in 53 (10.3%) and 62 (12.1%) cases respectively. A total of five cases (0.97%) coinfected with Pneumocystis and microsporidia were recovered from all analyzed autopsies. Coinfection of Pneumocystis and microsporidia is very challenging and raises interesting issues about host-parasite relationship. The early diagnosis of both pathogens must be crucial to establish correct and early treatments, improve the patient's evolution, reducing the risk of death.

Clinical analysis of 16 cases of pulmonary cryptococcosis in patients with normal immune function.

Pulmonary cryptococcosis (PC) is an invasive pulmonary fungal disease caused by Cryptococcus neoformans or Cryptococcus gattii. Cryptococcus is the opportunistic pathogen. Immune damage and chronic consumptive diseases are the high risk. The prevalence of cryptococcus infection is 5% to 10% in individuals with weakened immune function. In recent years, with the application of glucocorticoids, antitumor drugs, and broad-spectrum antibiotics, the prevalence of cryptococcal infection has increased yearly. Cryptococcus infection can also occur in normal people without underlying diseases. One in every 100,000 individuals with normal immune function is also affected, but these cases are often misdiagnosed due to inadequate understanding and low awareness of PC in individuals with normal immune function. In this study, we investigated the clinical characteristics of PC in patients with normal immune function to improve the diagnosis and treatment of these cases. We retrospectively analyzed the clinical, imaging and pathological data of 16 patients with pulmonary cryptococcosis treated at Beijing Shijitan Hospital between 2010 and 2017. Of the 16 patients with disseminated cryptococcosis, 6 were men, and 10 were women; their ages were 35 to 76 years (median: 53 years). Initial symptoms were cough, sputum, chest pain, and fever. Thirteen patients had a history of chronic disease or tumor, including diabetes and bronchial asthma (n=2), simple hypertension (n=2), chronic bronchitis (n=2), diabetes and hypertension (n=5), previous tuberculosis (n=1), and previous lung cancer surgery (n=1). Chest computed tomography (CT) in these 16 patients showed single or multiple nodules and masses (n=14), lobar consolidation (n=1), and diffuse mixed lesions (n =1). Lung biopsy showed granulomatous lesions in these 16 patients. Cryptococcus neoformans was identified with Grocott's methenamine silver and Grocott's methenamine silver (GMS) stains. Fourteen patients received fluconazole alone; two patients with intracranial infection received fluconazole combined with amphotericin B. Five patients improved significantly, and nine patients improved with significant lesion absorption. The clinical manifestations of PC are nonspecific in patients with normal immune function. Lung biopsy and cryptococcus antigen tests of cerebrospinal fluid are valuable diagnostic aids. Fluconazole is an effective treatment for disseminated cryptococcal infection.

Insights into myxozoan composition and physiology revealed by histochemical properties of myxospores.

Myxozoa (phylum Cnidaria) are a diverse group of metazoan parasites that predominately infect fish. Little is known regarding the composition and physiology of their myxospore life stage. The objective of this work was to investigate the composition of myxospores and extrasporogonic stages of nine myxozoan species infecting various teleost fish using histochemical staining techniques. Thirty histochemical stains were applied to formalin-fixed, paraffin-embedded tissues processed routinely for light microscopic evaluation. The polar capsules were the most consistent stain target across the taxa examined. Polar capsule staining with Alizarin red, von Kossa and methyl green-pyronin suggests the presence of intracapsular calcium and phosphate, which may contribute to polar filament discharge or pathogenesis of host invasion. The shell valves and suture lines of most myxozoans were stained with Luna and phosphotungstic acid haematoxylin stains, consistent with the presence of chitin and microfibrils, respectively. Vacuoles were consistently highlighted by diastase-susceptible periodic acid-Schiff and Grocott's methenamine silver staining, indicating glycogen. Other histochemical stains exhibited inconsistent staining across the taxa, suggesting differences in myxospore composition potentially reflective of physiologic variations and tissue tropisms. This work provides some information on conserved features and taxa-associated composition of myxospores and lends insight into myxozoan physiology and host-parasite interactions.

Nasal Septum Perforation Associated to Histoplasmosis: A Retrospective Study

Objective: To determine the prevalence of nasal septum perforation associated to Histoplasma sp. Material and Methods: A retrospective descriptive study was conducted with patients who developed nasal septum perforation associated to histoplasmosis. The diagnosis was made based on clinical manifestations and confirmed with mycological direct examination with Giemsa stain, culture, serologic tests (immunodiffusion method), and histopathology with hematoxylin-eosin, Grocott methenamine (GMS) and Periodic Acid-Schiff stain (PAS). Results: Out of 1654 medical records reviewed with diagnosis of histoplasmosis in the study period, we found 22 cases with nasal septum perforation. Autoimmune disease was present in 6 patients; all six were on immunosuppressive treatment. Two patients had HIV/AIDS, one of them had also paracoccidiodomycoses and the remaining had no apparent underlying disease or conditions . Conclusion: Physicians of endemic areas such as Venezuela, must consider this entity in the differential diagnosis with other diseases.

Different Vibratory Conditions Elicit Different Structural and Biological Vocal Fold Changes in an In-Vivo Rabbit Model of Phonation

Vibration of the vocal folds can disrupt the tissue and induce structural, functional, and molecular changes; the presence or absence of contact between the vocal folds during vibration can affect the type and extent of these changes. The purpose of this study was to characterize vocal fold changes following 2 hours of contact phonation or phonation without vibratory contact. Six New Zealand white breeder rabbits underwent 120 minutes of phonation with or without vibratory contact, and four served as nonphonated controls. The larynx was exposed and current was applied to the cricothyroids bilaterally to achieve vocal fold adduction while humidified airflow was delivered to induce vocal fold vibration. Laryngeal position, airflow, and stimulation levels were adjusted to obtain phonation with or without contact, and phonation was elicited for 120 minutes. Following excision, larynges were stained using Hematoxylin & Eosin, Elastica van Gieson, and Grocott's Methenamine Silver, or labeled with immunofluorescent markers for E-cadherin, CD31, CD11b, and Vimentin. All images were captured using a Nikon 90i microscope and analyzed using ImageJ. Differences between vibratory conditions and control samples were observed. There was more extensive epithelial thinning, reduced epithelial integrity and increased vascularity in the contact phonation group, while both phonatory groups demonstrated a decreased presence of mucous on the luminal surface and a decrease in elastin band thickness and lamina propria depth. Neither condition showed differences in inflammatory cell presence compared to control tissue. By showing that these two vibratory conditions result in structural changes of different types and magnitude, we have provided the first empirical evidence that vocal fold tissue is sensitive to differences in forces, and that changes in vibratory pattern can elicit different downstream biological changes within the tissue. The differences described herein are an important step toward understanding the vocal folds' potential for differential response to phonotraumatic damage following different vibratory behaviors.