MycoViro Lab Diagnosis Pt 2

Cheatsheet Content

### MycoViro Laboratory Diagnosis Part 2 This cheatsheet covers key aspects of laboratory diagnosis for mycology (fungi) and virology (viruses), focusing on methods and interpretations. #### Mycology: Fungal Infections #### Direct Microscopy (Mycology) - **Principle:** Rapid detection of fungal elements (hyphae, yeasts, spores) directly from clinical specimens. - **Specimens:** Skin scrapings, nail clippings, hair, sputum, CSF, tissue biopsies. - **Stains:** - **KOH Mount (Potassium Hydroxide):** Dissolves host cells, leaving fungal elements visible. Useful for skin, hair, nails. - **India Ink Stain:** Used for encapsulated yeasts like *Cryptococcus neoformans* in CSF, revealing a halo around the yeast. - **Calcofluor White:** Fluorescent stain that binds to chitin in fungal cell walls. Requires UV microscope. Highly sensitive. - **Gram Stain:** Fungi are Gram-positive (purple), but morphology is more critical than Gram reaction. Useful for yeast-like fungi. - **Lactophenol Cotton Blue (LPCB):** Stains fungal elements blue, useful for visualizing morphology in cultures. #### Fungal Culture - **Principle:** Gold standard for identifying fungi. Allows for isolation, identification, and susceptibility testing. - **Media:** - **Sabouraud Dextrose Agar (SDA):** General purpose, acidic pH inhibits bacteria. Contains glucose and peptone. - **Mycosel Agar:** SDA with cycloheximide (inhibits saprophytic fungi) and chloramphenicol (inhibits bacteria). Selective for dermatophytes. - **Brain Heart Infusion (BHI) Agar:** Enriched medium for fastidious fungi, especially dimorphic fungi. - **Chromogenic Media:** Differentiate common yeasts (e.g., *Candida* species) by color production. - **Incubation:** Typically 25-30°C for dermatophytes/molds, and 35-37°C for yeasts and dimorphic fungi (yeast phase). Incubate for up to 4 weeks or longer. - **Identification:** Based on macroscopic (colony morphology, texture, color) and microscopic (hyphae, spores, budding) characteristics. #### Molecular Methods (Mycology) - **PCR (Polymerase Chain Reaction):** Amplifies specific fungal DNA sequences. - **Advantages:** Rapid, sensitive, can detect non-viable fungi. Useful for difficult-to-culture fungi or when rapid diagnosis is critical (e.g., invasive aspergillosis). - **Targets:** Ribosomal RNA genes (e.g., 18S, 28S, ITS regions) are common. - **FISH (Fluorescence In Situ Hybridization):** Uses fluorescently labeled probes to detect specific fungal species directly in clinical specimens. - **MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry):** Identifies fungi based on their unique protein profiles. Rapid and accurate for identifying cultured isolates. #### Serological Tests (Mycology) - **Principle:** Detects fungal antigens or host antibodies against fungi. - **Antigen Detection:** - **Galactomannan:** Polysaccharide in *Aspergillus* cell wall. Detected in serum, BAL fluid for invasive aspergillosis. - **(1,3)-β-D-Glucan:** Pan-fungal cell wall component, detected in serum for various invasive fungal infections (e.g., *Candida*, *Aspergillus*, *Pneumocystis*). Not specific for a single fungus. - **Cryptococcal Antigen:** Detected in CSF and serum for cryptococcosis. Latex agglutination or EIA. - **Histoplasma Antigen:** Detected in urine, serum, BAL fluid for histoplasmosis. - **Antibody Detection:** Less useful for acute invasive infections due to delayed antibody response and presence in healthy individuals (exposure). More useful for chronic infections (e.g., coccidioidomycosis, blastomycosis). #### Antifungal Susceptibility Testing - **Methods:** Microbroth dilution, disk diffusion, Etest. - **Indications:** Invasive infections, recurrent infections, infections in immunocompromised patients, or when resistance is suspected. - **Commonly tested fungi:** *Candida* species, *Aspergillus* species. #### Virology: Viral Infections #### Direct Detection (Virology) - **Principle:** Detects viral components (antigens, nucleic acids) or viral particles directly from specimens. - **Electron Microscopy (EM):** Visualizes viral particles. Rarely used for routine diagnosis due to cost and expertise, but useful for identifying unculturable viruses or in outbreak investigations. - **Immunofluorescence (DFA/IFA):** Detects viral antigens in infected cells using fluorescently labeled antibodies. Rapid, useful for respiratory viruses (e.g., influenza, RSV). - **Enzyme-linked Immunosorbent Assay (ELISA):** Detects viral antigens (e.g., HIV p24 antigen, HBsAg). Can be quantitative. - **Rapid Antigen Tests:** Lateral flow immunoassays for quick detection in point-of-care settings (e.g., influenza, SARS-CoV-2). Lower sensitivity than PCR. #### Molecular Methods (Virology) - **PCR (Polymerase Chain Reaction):** Amplifies viral nucleic acids (DNA or RNA). - **RT-PCR (Reverse Transcriptase PCR):** For RNA viruses (e.g., influenza, HIV, SARS-CoV-2). RNA is first converted to cDNA. - **Real-time PCR (qPCR):** Quantifies viral load, faster, and reduces contamination risk. Used for HIV, HBV, HCV viral load monitoring. - **Multiplex PCR:** Detects multiple viruses simultaneously (e.g., respiratory panels). - **Advantages:** High sensitivity, specificity, rapid, can detect non-viable viruses. - **Next-Generation Sequencing (NGS):** - **Principle:** Sequences entire viral genomes. - **Applications:** Outbreak investigation, detection of novel viruses, drug resistance profiling, quasispecies analysis. #### Viral Culture - **Principle:** Inoculating clinical specimens into cell lines to grow and isolate viruses. - **Cell Lines:** Vary depending on the virus (e.g., HEp-2 for RSV, Vero for HSV, MDCK for influenza). - **Detection of Viral Growth:** - **Cytopathic Effect (CPE):** Visible changes in infected cells (e.g., rounding, lysis, syncytia formation). - **Hemadsorption:** Some viruses cause infected cells to adsorb red blood cells. - **Immunofluorescence/ELISA:** Confirms viral identity using specific antibodies. - **Disadvantages:** Time-consuming (days to weeks), requires viable virus, not all viruses can be cultured. Less common for routine diagnosis due to molecular methods. #### Serological Tests (Virology) - **Principle:** Detects host antibodies (IgM, IgG) against viral antigens. - **IgM Antibodies:** Indicate recent or acute infection. - **IgG Antibodies:** Indicate past infection, immunity, or chronic infection. Rise in IgG titer between acute and convalescent sera (four-fold increase) is diagnostic of acute infection. - **Methods:** ELISA, Western Blot (confirmatory for HIV), Immunofluorescence. - **Applications:** Diagnosis of acute infection, determination of immune status (e.g., measles, rubella), screening for chronic infections (e.g., HIV, HBV, HCV). - **Limitations:** Window period (before antibody production), cross-reactivity, maternal antibodies in neonates. #### Antiviral Susceptibility Testing - **Methods:** Cell culture-based assays, genotypic assays (sequencing for resistance mutations). - **Indications:** Chronic infections (e.g., HIV, HBV, HCV) to guide therapy, suspected resistance in immunocompromised patients.