fungal sinusitis, clasificationt and pathogenesis

Supplement about fungal sinusitis, clasificationt, management therapy and pathogenesis

1. SUPPLEMENT

2. CLSI M38-A3 standard for moulds : Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard.  CLSI document M38-A3. CLSI, Pennsylvania, USA 2017. The M38-A3 standard describes a method for testing antifungal susceptibility of filamentous fungi ( moulds ) that cause invasive infections, including  Aspergillus  spp.,  Fusarium  spp.,  Scedosporium  spp., zygomycetes and other pathogenic moulds . It recommends the use of RPMI-1640 medium (with glutamine, without bicarbonate, and with phenol red as a pH indicator. Microdilution trays are incubated at 35oC; and may be read at 24 hours for Rhizopus species; 48 hours for Aspergillus, Fusarium and  Sporothrix  species; and 72 hours for slower growing moulds like  Scedosporium / Lomentospora  species. Most moulds may be read at 48 hours. Turbidity in the microdilution wells should be scored with the aid of a reading mirror and compared with that of the growth control.  A numerical score from 0 to 4 is given to each well using the following scale:  0 = optically clear or absence of growth 1 = slight growth (25% of growth control) 2 = prominent reduction in growth (50% of growth control) 3 = slight reduction in growth (75% of growth control) 4 = no reduction in growth https://www.adelaide.edu.au/mycology/laboratory-methods#antifungal-susceptibility-testing

3. Characteristics CLSI M38-A3   EUCAST Edef 9.3.1 Format Round based wells Flat based wells Final Inoculum 0.4 - 5 x 10 4  CFU/mL 1 - 2.5 x 10 5  CFU/mL Test medium RPMI 1640 with 0.2% glucose RPMI 1640 with 0.2% glucose Growth Temperature  35°C  35°C Incubation time 48 h (21-74 h) 48 h (24-72 h) Endpoint 100% inhibition Except: aberrant growth (MEC) for echinocandins 100% inhibition Except: aberrant growth (MEC) for echinocandins Reading Visually  Visually Essential differences between CLSI and EUCAST broth microdilution standard methodologies for moulds . https://www.adelaide.edu.au/mycology/laboratory-methods#antifungal-susceptibility-testing

4. Reading MICs Skipped wells may occur due to (a) inconsistent inoculum dispensed into wells, (b) poor growth habit of organism, and (c) a manufacturing error when the drug as dispensed into wells.  Repeat the test if two or more skipped wells are present. Some azoles, particularly fluconazole, exhibit a phenomenon known as trailing. Trailing occurs when the turbidity continually decreases as the drug concentration increases but the suspension fails to become optically clear (partial inhibition of growth over an extended range of antifungal concentrations). For most isolates, the difference between reading at 24 hours versus 48 hours is minimal and will not alter the interpretative category (i.e. does not change whether the isolate would be read as “susceptible” or “resistant”). However some isolates show a dramatic rise in MIC over time (e.g. for fluconazole from 0.5 μg /ml at 24 hours to 256 μg /ml at 48 hours). This trailing phenomenon has been reported as occurring in about 5% of isolates,8 however some studies have reported that up to 20% of C. albicans isolates read at 48 hours may show trailing to fluconazole that would alter the interpretation from “susceptible” to “resistant” 6,7. Summary: The main practical difficulties with antifungal susceptibility testing are usually with the inoculum preparation and endpoint determination. An inoculum sterility and density check should be done for each test [i.e. spread 10 μL of the inoculum onto a clean SDA plate and count the CFU’s at 24 hours]. This also acts as a growth control for slower growing fungi [i.e. you may have to read some plates at 48 or 72 hours]. If the inoculum is too light or heavy the test should be repeated. Endpoint reading may also be enhanced by the use of colorimetric indicators, plate readers and automated zone readers, which allow for a more quantitative result.  https://www.adelaide.edu.au/mycology/laboratory-methods#antifungal-susceptibility-testing

5. Galleria mellonella , also known as the wax moth, belongs to the  Pyralidae  family in the Lepidopteran order. Duration of its life cycle can vary from weeks to month depending on several factors, especially food, temperature, and humidity. Kelompok homogen yang terdiri dari 10 hingga 20 larva dengan kutikula berwarna krem , dengan berat sekitar 200–300 mg, panjang 1–3 cm dan mobilitas spontan , umumnya digunakan . Larva harus dimanipulasi dengan hati-hati untuk menghindari tekanan fisik .  Setelah terinfeksi , larva dipelihara hingga suhu 37°C tanpa diberi makan .  Pergerakan larva secara bertahap menurun , mencerminkan perkembangan infeksi jamur , namun pada tingkat yang bervariasi tergantung pada spesies dan strain jamur . Evaluasi morbiditas memberikan rincian lebih lanjut mengenai perkembangan infeksi pada larva.  Sistem penilaian yang terdiri dari empat kriteria utama , melanisasi , mobilitas , kapasitas membentuk kepompong sutra, dan kelangsungan hidup , telah digunakan dalam beberapa penelitian perkembangan aspergillosis invasif pada larva menunjukkan kemiripan dengan yang terjadi pada mamalia . Sheehan  dkk  .  menunjukkan bahwa inokulasi konidia diikuti oleh (i) pembentukan nodul melanisasi dan (ii) peningkatan kepadatan hemosit dan peptida antimikroba . Larva yang terinfeksi mengalami melanisasi seiring berjalannya waktu dengan membentuk kapsul melanisasi yang mengelilingi patogen , dan organ dalamnya menjadi tidak teratur akibat infeksi . Marie-Fleur Durieux, Élise Melloul , Sana Jemel , Lolita Roisin, Marie-Laure Dardé , Jacques Guillot, Éric Dannaoui & Françoise Botterel (2021) Galleria mellonella as a screening tool to study virulence factors of Aspergillus fumigatus, Virulence, 12:1, 818-834, DOI: 10.1080/21505594.2021.1893945 Anatomy of a larva of  Galleria mellonella , adapted from Singkum   et al ., 2018 and Engel and Moran, 2013

6. Beth Burgwyn Fuchs, Elizabeth O’Brien, Joseph B. El Khoury & Eleftherios Mylonakis (2010) Methods for using Galleria mellonella as a model host to study fungal pathogenesis , Virulence, 1:6, 475-482, DOI: 10.4161/viru.1.6.12985

7. Calmodulin Calmodulin adalah protein pengikat Ca2+ yang terdapat di semua sel eukariotik yang berfungsi sebagai reseptor intraseluler utama untuk Ca2+.  Protein 148 asam amino ini terlibat dalam aktivasi lebih dari 20 enzim yang memediasi berbagai proses fisiologis .  Banyak dari enzim ini dihambat secara intramolekul dan kompleks Ca(2+)-calmodulin mengurangi penghambatan ini .  Calmodulin sangat penting bagi kehidupan karena gangguan gen pada organisme yang dapat diatur secara genetis dapat berakibat fatal.  Protein ini memainkan peran pengaturan yang penting dalam proliferasi sel dan diperlukan di berbagai titik dalam siklus sel.  Mekanisme aktivasi enzim oleh calmodulin dan pentingnya dalam regulasi pertumbuhan sel ditinjau .

8. Apa saja penerapan analisis filogenetik ? Analisis filogenetik memberikan pemahaman mendalam tentang bagaimana spesies berevolusi melalui perubahan genetik .  Dengan menggunakan filogenetik , para ilmuwan dapat mengevaluasi jalur yang menghubungkan organisme masa kini dengan asal usul leluhurnya , serta memprediksi perbedaan genetik yang mungkin terjadi di masa depan . Filogenetik memiliki banyak penerapan dalam bidang medis dan biologi , termasuk ilmu forensik , biologi konservasi , epidemiologi , penemuan obat dan desain obat , prediksi struktur dan fungsi protein, serta prediksi fungsi gen. Estimasi yang lebih akurat mengenai hubungan evolusi antar spesies kini dapat dilakukan melalui analisis filogenetik molekuler menggunakan data pengurutan gen.  Selain itu , klasifikasi Linnaean ( berdasarkan keterkaitan ciri-ciri fisik yang jelas ) dari spesies yang baru berevolusi dapat dilakukan dengan menggunakan analisis filogenetik molekuler .   Mengenai aplikasi kesehatan masyarakat , analisis filogenetik molekuler dapat digunakan untuk mengumpulkan informasi tentang wabah patogen .  Kemungkinan sumber penularan patogen dapat diselidiki dengan menganalisis hubungan epidemiologis antara rangkaian genetik suatu patogen , seperti HIV. Analisis filogenetik dapat berguna dalam genomik komparatif , yang mempelajari hubungan antara genom spesies berbeda .  Dalam konteks ini , salah satu penerapan utamanya adalah prediksi gen atau penemuan gen, yang berarti menemukan lokasi wilayah genetik tertentu di sepanjang genom . Skrining filogenetik terhadap spesies yang berkerabat secara farmakologis dapat membantu mengidentifikasi anggota spesies yang berkerabat dekat dengan signifikansi farmakologis . Dalam mikrobiologi , analisis filogenetik dapat diterapkan untuk mengidentifikasi dan mengklasifikasikan berbagai mikroorganisme , termasuk bakteri . Selain itu , filogenetik dapat digunakan untuk mengevaluasi interaksi evolusi timbal balik antara mikroorganisme , serta untuk mengidentifikasi mekanisme (transfer gen horizontal) yang bertanggung jawab atas adaptasi cepat patogen dalam lingkungan mikro inang yang selalu berubah .     

9. Inhalation of aflatoxins has been associated with occupations involving exposure to environmental molds 24 , such as grain processing. However, aflatoxins can increase protein kinase C (PKC) activity in some cell lines  in vitro 29 , 30 , 31 . Because PKC can decrease ciliary beat frequency (CBF) 32 , 33   we hypothesized that aflatoxins may have acute effects on Mucociliary clearance (MCC) that contribute to  A. flavus  pathogenesis.

10. Applications of DNA Sequencing

11. Current understanding of the pathogenesis of invasive aspergillosis: alveolar macrophages and monocytes form the first line of host defense after inhalation of Aspergillus conidia from the environment the host innate immune defense is activated, resulting in phagocytic ingestion of conidia and release of microbicidal compounds In the absence of adequate immune containment, conidial germination with development of hyphae heralds invasive disease. Neutrophils, which form the second line of defense, attack the hyphae through release of oxidants and degranulation. These are lacking in the neutropenic host. Meanwhile, antigen-presenting functions, as performed by mononuclear phagocytes and dendritic cells, facilitate the development of a T helper (Th) cell–based adaptive immune response During this process of immune activation and response, cytokines play a pivotal role in mediating the host inflammatory response. Interleukin (IL)–6 is a pleiotropic cytokine produced by macrophages and lymphocytes. It facilitates lymphocyte differentiation and proliferation of hematopoietic progenitor cells and is an important mediator of the acute-phase response of which C-reactive protein (CRP) is a major product. IL-8, released by monocytes, macrophages, and endothelial cells, recruits neutrophils and induces chemotaxis to the site of infection. T cell–derived cytokines also play an important role in the modulation of host defense. Interferon (IFN)–g is the prototypic type 1 T helper lymphocyte (Th1) proinflammatory cytokine that activates cell-mediated immune responses, whereas IL-10 is an anti-inflammatory product mainly released by type 2 T helper lymphocytes (Th2) and T regulatory (Treg) lymphocytes that keeps in check the inflammatory cascades.