Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

Chromosome-Level Genome Assembly of a Human Fungal Pathogen Reveals Synteny among Geographically Distinct Species

Histoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa, and Europe.

Variation in Host Resistance to Blastomyces dermatitidis: Potential Use of Genetic Reference Panels and Advances in Immunophenotyping of Diverse Mouse Strains

Host genetic variation significantly impacts vulnerability to infectious diseases. While host variation in susceptibility to fungal infection with dimorphic fungi has long been recognized, genes that underpin this variation are poorly understood.

Two Novel Dimorphism-Related Virulence Factors of Zymoseptoria tritici Identified Using Agrobacterium-Mediated Insertional Mutagenesis

Diseases caused by dimorphic phytopathogenic and systemic dimorphic fungi have markedly increased in prevalence in the last decades, and understanding the morphogenic transition to the virulent state might yield novel means of controlling dimorphic fungi. The dimorphic fungus Z. tritici causes significant economic impact on wheat production, and yet the regulation of the dimorphic switch, a key first step in successful plant colonization, is still largely unexplored in this fungus. The fungus is amenable to suppression by fungicides at this switch point, and the identification of the factors controlling the dimorphic switch provides a potential source of novel targets to control Septoria tritici blotch (STB). Inhibition of the dimorphic switch can potentially prevent penetration and avoid any damage to the host plant. The aim of the current work was to unveil genetic determinants of the dimorphic transition in Z. tritici by using a forward genetics strategy. Using this approach, we unveiled two novel factors involved in the switch to the pathogenic state and used reverse genetics and complementation to confirm the role of the novel virulence factors and further gained insight into the role of these genes, using transcriptome analysis via RNA-Seq. The transcriptomes generated potentially contain key determinants of the dimorphic transition.

Pandora’s Box: Disseminated Coccidioidomycosis Associated with Self-Medication with an Unregulated Potent Corticosteroid Acquired in Mexico

Coccidioidomycosis (CM), caused by the dimorphic fungi Coccidioides immitis and C. posadasii, typically presents as acute or chronic pulmonary disease. However, disseminated disease occurs in about 1% of patients. Disseminated CM may affect multiple organ systems, including cutaneous, osteoarticular, and central nervous system sites. Here, we present a case of disseminated CM in a patient from a border city in Texas. The patient had a history of uncontrolled diabetes mellitus and was also taking an over-the-counter medication acquired in Mexico that contained a potent corticosteroid. The patient presented with seizures and was found to have a brain infarct, cavitary lung lesions, synovitis of the knee, multiple skin lesions, and chorioretinitis. The patient had a very high complement fixation titer for Coccidioides; fungal spherules were seen in a skin biopsy specimen, and Coccidioides grew in culture from a sample of synovial fluid and the skin biopsy specimen. This case illustrates the dissemination potential of Coccidioides, the danger of unregulated pharmaceuticals, the importance of thorough history taking, and recognizing risk factors that contribute to disseminated CM.

Fungal Infections of the Central Nervous System

Fungal infections within the central nervous system (CNS) occur more commonly in patients with immunosuppressive conditions but occasionally are diagnosed in immunocompetent patients as well. This may reflect the route of infection and the specific pathogen. Fungal infections resulting from hematogenous seeding are typically encountered in patients with profound and prolonged immunosuppression (including neutropenia); however, pathogens such as Cryptococcus gattii and some dimorphic fungi can also produce CNS disease in otherwise healthy persons. A common avenue of infection in immunocompetent patients is traumatic or nosocomial inoculation, including neurosurgical procedures, foreign body implants, and contaminated spinal fluid injections.

708. Evaluation of Fungal Culture versus Bacterial Culture for the Identification of Various Mold Species

Background Invasive mold infections are challenging to diagnose and in part relies on fungal cultures. A large proportion of mold isolates are recovered on routine bacterial cultures in our medical center, thus we sought to define the utility of bacterial versus fungal cultures for isolation of mold from clinical specimens. Methods Routine bacterial and fungal culture results from wound, tissue, body fluid, and respiratory specimens from Jan 2019-Dec 2020 from Keck Medical Center of USC (Los Angeles, CA) were retrospectively reviewed. Cases were excluded if specimens were collected specifically for dermatophyte recovery or for blood culture. Cultures in which mold, including dimorphic fungi, were isolated were included in the evaluation. Results Mold was isolated from 612 specimens from 408 patients, with recovery from 329 bacterial and 450 fungal cultures. Among the 329 bacterial cultures, fungal cultures were not requested in 119 (36.2%) while the remaining 210 had concurrent fungal cultures which recovered mold in 167 cases (79.5%). Of 450 fungal cultures recovering mold, a corresponding bacterial culture was performed in 445, isolating mold in 181 (38.8%) of these cases. Two or more molds were found in 28 fungal cultures and in 5 bacterial cultures. Of positive specimens with both fungal and bacterial cultures performed (n=488), mold was isolated in fungal cultures in 446 (91.4%) and in bacterial cultures in 209 (42.9%) (Table). Yield of molds in 488 specimens with concomitant bacterial and fungal cultures Conclusion Although a significant number of molds are recovered in routine bacterial cultures, over half would be missed without concomitant fungal cultures. Conversely, recovery of clinically relevant mold species was optimal when both bacterial and fungal cultures were requested on a specimen. This may be related to increased specimen sampling and incubation conditions allowing for broader organism recovery. Disclosures All Authors: No reported disclosures

Caenorhabditis elegans as an Infection Model for Pathogenic Mold and Dimorphic Fungi: Applications and Challenges

The threat burden from pathogenic fungi is universal and increasing with alarming high mortality and morbidity rates from invasive fungal infections. Understanding the virulence factors of these fungi, screening effective antifungal agents and exploring appropriate treatment approaches in in vivo modeling organisms are vital research projects for controlling mycoses. Caenorhabditis elegans has been proven to be a valuable tool in studies of most clinically relevant dimorphic fungi, helping to identify a number of virulence factors and immune-regulators and screen effective antifungal agents without cytotoxic effects. However, little has been achieved and reported with regard to pathogenic filamentous fungi (molds) in the nematode model. In this review, we have summarized the enormous breakthrough of applying a C. elegans infection model for dimorphic fungi studies and the very few reports for filamentous fungi. We have also identified and discussed the challenges in C. elegans-mold modeling applications as well as the possible approaches to conquer these challenges from our practical knowledge in C. elegans-Aspergillus fumigatus model.

Detection of Coccidioides posadasii in a patient with meningitis using metagenomic next-generation sequencing: a case report

Background Coccidioidomycosis is a systemic infection caused by dimorphic fungi Coccidioides spp. endemic to Southwestern United States and Central and South America. A history of residence and travel in these areas is essential for the diagnostic of coccidioidomycosis, which has highly variable symptoms ranging from asymptomatic to severe, disseminated infection, and even death. Immunocompromised patients of coccidioidomycosis experience a high risk of dissemination, chronic infection, and mortality. Meningitis is one of the most deleterious coccidioidomycosis and can cause various life-threatening complications. Case presentation Here we report a case of Coccidioides posadasii meningitis in a 49-year-old female who returned to China after one and a half years residence in Los Angeles, USA. The repeated routine cultures using CSF for bacteria or fungi were all negative. To hunt for an infectious etiology, the state-of-the-art technology metagenomic next-generation sequencing (mNGS) was then utilized, suggesting Coccidioides posadasii. Organizational pathological examination and polymerase-chain-reaction (PCR) results subsequently confirmed the mNGS detection. Conclusion To our knowledge, cases for coccidioidal meningitis have been rarely reported in China. While global travelling may spread this disease across continents and make the diagnosis more difficult. mNGS can detect almost all known pathogens with high sensitivity and specificity, especially for uncommon pathogen, such as Coccidioides posadasii in China.

Transcriptional regulatory features associated with Coccidioides immitis phase transition

Coccidioidomycosis (Valley Fever) is an emerging endemic fungal infection with a rising incidence and an expanding geographic range. It is caused by Coccidiodes, which are thermally dimorphic fungi that grow as mycelia in soil but transition in the lung to form pathogenic spherules. The regulatory mechanisms underlying this transition are not understood. Exploiting capped small (cs)RNA-seq, which identifies actively initiated stable and unstable transcripts and thereby detects acute changes in gene regulation with remarkable sensitivity, here we report the changes in architectural organization and key sequence features underlying phase transition of this highly pathogenic fungus. Spherule transition was accompanied by large-scale transcriptional reprogramming, functional changes in transcript isoforms, and a massive increase in promoter-distal transcription of ncRNAs. Analysis of spherule-activated regulatory elements revealed a motif predicted to recruit a WOPR family transcription factor, which are known regulators of virulence in other fungi. We identify CIMG_02671 as a C. immitis WOPR homologue and show that it activates transcription in a WOPR motif-dependent manner, suggesting it is an important regulator of pathogenic phase transition. Collectively, this also highlights csRNA-seq as a powerful means to identify transcriptional mechanisms that control pathogenesis.