Mycology and Virology M1. Systemic Mycoses 🍄 🦠 PDF

| Topic 6 | [MLS 415] Mycology and Virology M1. Systemic Mycoses Professor: Joanne Krystianne Tago Date: March 3, 2024 Primary Systemic Mycoses ENDEMIC MYCOSES → particularly “Primary” Systemic Mycoses – to differentiate it from other pathogenic fungal elements because some species can also cause di...

| Topic 6 | [MLS 415] Mycology and Virology M1. Systemic Mycoses Professor: Joanne Krystianne Tago Date: March 3, 2024 Primary Systemic Mycoses ENDEMIC MYCOSES → particularly “Primary” Systemic Mycoses – to differentiate it from other pathogenic fungal elements because some species can also cause disseminated systemic infections Coccidioidomycosis Histoplasmosis Blastomycosis Paracoccidioidomycosis Caused by DIMORPHIC FUNGI YEAST PHASE On Enriched media w/ Blood, 35 – 37 deg C Seen in vivo, aka “tissue/invasive phase” Coccidioidomycosis MYCELIAL PHASE On Sabouraud Dextrose Agar at 25 – 30 deg C Saprophytic, observed in vitro Transmission Inhalation of Fungal Spores (Most common) Initial: Pulmonary Infection (Symptomatic/Asymptomatic) o symptomatic/asymptomatic o if untreated, it will develop into a disseminated forms of infection or progressive infections o have severe symptoms brought about by the spread of organisms from the lungs to distant organs Dissemination (in HIV or prolonged corticosteroid treatment) o mostly seen in immunocompromised individuals − HIV or under prolonged corticosteroid treatment Geographic Restrictions o Endemic because causative agents have a specific location where they thrive Serological Tests: o (1) Complement Fixation Test, (2) Immunodiffusion Test, (3) Tube precipitin Test, (4) Enzyme Immunoassay Most infections are asymptomatic or mild and resolve without treatment however, a small but significant number of patients develop pulmonary disease, which may involve dissemination from the lungs to other organs - (Arthroconidia of Coccidioides spp.) Also known as: o Valley fever o San Joaquin Valley Fever o Desert fever/ desert rheumatism MOT: Inhalation of arthroconidia o some cases were caused by puncture with infected objects or organ transplantation, and sexual contact (very rare) 60% = asymptomatic and self-limited Respiratory Tract Infections (Males are more susceptible) 40% = Flu-like condition DISSEMINATION Visceral organs Meninges Bone Skin Lymph nodes Subcutaneous tissue The disease occurs more frequently in certain racial groups (commonly on dark-skinned individual); in decreasing order: 1. Filipinos, 2. African Americans, 3. Native Americans, 4. Hispanics, and 5. Asians Biology of Coccidioidomycosis The initial host defenses are provided by the alveolar macrophages, which are usually capable of inactivating the conidia and inducing a robust immune response this process typically leads to granulomatous inflammation and the production of both antibodies and cell-mediated immunity Source: Centers for Disease Control and Prevention Found in Soil or Dusty environment @mlstranses | 1 In the environment (Desserts), Coccidioides ssp. exists as a: 1. Mold with septate hyphae 2. The hyphae fragment into arthroconidia which measure only 2-4 um in diameter. 3. Arthroconidia are EASILY AEROSOLIZED when disturbed. 4. Arthroconidia are inhaled by a susceptible host (4) and settle into the lungs. 5. The new environment (37 deg C) signals a morphologic change, and the arthroconidia become SPHERULES. 6. Spherules divide internally until they are filled with endospores (6). 7. When a spherule ruptures (7) the endospores are released and disseminate within surrounding tissue. 8. ENDOSPORES are then able to develop into NEW SPHERULES and repeat the cycle. Coccidioides spp. BOTH ARE MORPHOLOGICALLY INDISTINGUISHABLE o Coccidioides immitis = “California” spp. o Coccidioides posadasii = “Non-California” spp. ▪ Common in Mexico ▪ Slow growing on media with high salt concentration (Halophilic) DISSEMINATED COCCIDIOIDES Life Cycle of Coccidioides (Source: Barker, n.d., Obtained from ResearchGate) When a conidia lands a favorable environment, it will form into hyphal fragments 2. these hyphal fragments will branch out and entangle producing mycelia 3. the hyphal fragments in the mycelia will produce arthroconidia, which will break to produce the single arthroconidia 4. When the soil that contains the arthroconidia is disturbed, the arthroconidia may become dispersed in the air 5. when accidentally inhaled by an individual, the arthroconidia will morphologically change into its tissue or invasive phase due to temperature change ⇒ spherule 6. sporangiospores or endospores are then formed within the spherule by repeated cytoplasmic cleavage 7. the spherule will become engorged 8. after 120 hours, the spherules rupture releasing 9. endospores 10. the endospores may either be: a. released outside the body and proceed with saprobic life cycle b. remains within the body and reinitiate the cycle of spherules development 1. [L]: Chronic cutaneous coccidioidomycosis ; [R]: Extension of Pulmonary Coccidioidomycosis (Armpits) Begins with pulmonary infection → Severe cutaneous involvement (Granulomatous lesions) It may also show a large superficial ulcerated plaques under the armpits and upper chest; involvement of muscles and subcutaneous tissue and skin after pulmonary infection ○ There is also a presence of draining sinuses (Holes seen in the R image) Coccidioides immitis – LABORATORY DIAGNOSIS 1. Blood testing, PCR, Chest X-ray 2. Direct Examination a. Finding of spherules in sputum, draining sinuses or tissue specimen b. 10-20% KOH; Calcofluor White Microscopic Characteristics of Coccidioides immitis MYCELIAL PHASE YEAST/TISSUE FORM Septate, branched hyphae Non-budding, thick walled Thick-walled barrel-shaped, spherule rectangular arthroconidia that Outer wall: Mannan, protein, alternate with empty cells called lipid disjuncture cells Inner wall: Chitin, 3-O-Methyl mannose 20-200 um in size @mlstranses | 2 With numerous non-budding spores small, Confirmatory Tests for Coccidioidomycosis IMMUNODIFFUSION TEST POSITIVE RXN: Precipitin line/band formation Detects coccidioidal IgM (precipitins) & IgG (CF) Indicates ACTIVE primary infection Macroscopic Characteristics of Coccidioides immitis Control 1 IgM-positive serum Control 2 IgG-positive serum Antigen 1 antigen reactive to IgM antibodies Antigen 2 antigen reactive to both IgM and IgG antibodies Observe rxn after 24 hours; if the control has no lines observed repeat the test Serological Test for Coccidioidomycosis: COMPLEMENT FIXATION TEST 3-21 days Sabouraud Dextrose Agar (SDA), Inhibitory Mold Agar (IMA) Delicate, fluffy white, which turns tan or brown with age. In vitro production of spherules: Incubation in a complex medium at 40 degrees Celsius with 20% Carbon dioxide (CO2) or by animal inoculation; to shift from mold to yeast CAUTION for Coccidioides immitis The MOST INFECTIOUS of all fungi Formerly a “Select agent” – biological agent that has the potential to pose a severe threat to public health and safety HANDLING: Use a Biosafety Cabinet for all preparation and handling Cultures MUST be sealed in a tape Cotton-plugged tubes is DISCOURAGED o Screw-capped tubes is needed Autoclave all cultures after identification is done! 1. 2. Skin Tests for Coccidioidomycosis Coccidioidin a. Crude toluene extract of mycelial culture i. rgnt contains arthroconidia b. (+) Induration of less than 5 mm Spherulin a. Prepared from cultured spherules b. More sensitive, but less specific i. not specific because there are other organism that produces spherules (e.g. R. seeberi) Sample: Patient’s serum Reagents: commercially known coccidioidin antigen commercially known complement sheep’s RBCs coated with antibodies (indicator) This test relies on two intrinsic properties of complement complement will lyse sensitized RBCs complement will bind to the Fc portion of the IgG antibodies bound to antigens Result absence of hemolysis presence of hemolysis Interpretation POSITIVE (+) NEGATIVE (-) Treatment for Coccidioidomycosis SUPPORTIVE CARE o Self-limiting, mild symptoms MODERATE CASES o Fluconazole or Itraconazole SEVERE CASES o Posaconazole or Amphotericin B o Treatment for the lesions @mlstranses | 3 1. Histoplasmosis Most prevalent pulmonary mycosis MOT: Inhalation of conidia or small hyphal fragments Chronic granulomatous infection Begins in the lungs with Tuberculosis-like infections o Resembles S/s of TB Mostly asymptomatic and self-limited May disseminate and Re-infection may occur Sources of Infection: Histoplamosis Silos, air-conditioning units contaminated with BIRD DROPPINGS o birds are not infected, but their excrement provides superb culture conditions for growth of the fungus Accumulations of guano (debris of bats) in caves, attics, or parks Nitrogen-rich soils (where they usually thrive) o bat and bird poop increases the nitrogen levels in soil Clinical Manifestations of Histoplasmosis ACUTE PULOMARY HISTOPLASMOSIS b. usually seen on immunocompromised individuals OCULAR HISTOPLASMOSIS a. b. c. Loss of central vision – retina is the most affected Initial symptoms may be loss of central vision, blind spots, or scars on the retina Late stages – abnormal blood vessels in the eye may develop and cause changes in the vision Histoplasma capsulatum as the causative agent of Histoplasmosis a. b. 2. Self-limiting Fever, malaise, cough, headache, pain chills, and myalgias c. miliary: presence of grain-like nodules CHRONIC PULMONARY HISTOPLASMOSIS 4. 1. 2. 3. 4. 5. 6. a. b. 3. Opportunistic complication of Chronic Obstructive Pulmonary Disease (COPD) with emphysema and abnormal pulmonary spaces Silent killer – it would start really early and only discovered after 5-10 years DISSEMINATED HISTOPLASMOSIS In the environment (Caves, attics, silos), Histoplasma capsulatum exists as a mold with aerial hyphae. The hyphae produce macroconidia and microconidia spores that are aerosolized and dispersed. Microconidia are inhaled into the lungs by a susceptible host. The warmer temperature inside the host signals a transformation to an oval, budding yeast. The yeast are phagocytized by immune cells and transported to regional lymph nodes. From there they travel in the blood to other parts of the body (through the circulatory system) Laboratory Diagnosis of Histoplasmosis Direct Microscopic Examination Difficult to see in the sputum and other tissues Rarely in peripheral blood Bone marrow smear: Wright or Giemsa (BEST) Intracellular yeast in macropahges Dermatomyositis-like pattern; tubules appear like tuberculoid form of leprosy a. Yeast cells may be disseminated while inside macrophages @mlstranses | 4 Macroscopic Characteristics of Histoplasma capsulatum Microscopic Characteristics of Histoplasma capsulatum MYCELIAL PHASE YEAST PHASE Septate hyphae 2-5 u (microns) Large spherical or From a mold tuberculate Brain-Heart Infusion macroconidia Agar at 37 degrees Small, round, smooth Celsius microconidia Small, ellipsoidal Slow-growing (2-4 weeks) Sabouraud Dextrose Agar/ Inhibitory Mold Agar (MOLD): ○ White → brown, fine fluffy; white, yellow, or tan on reverse side when cultured, demonstrates 2 colonial forms ○ colony A: albino-type (white) ○ colony B: slightly tinged/colored (brown or cream or tan) both colony types can form identical yeast and tissue forms − but colony B is more pathogenic for mice and rabbits and can produce more macroconidia Brain Heart Infusion Agar (YEAST): Moist, white to cream heaped colony Histoplasmin Skin Test Produced by growing the mycelium in asparagine broth medium Positive Reaction: indicated by an area of induration >5mm in diameter after 48 hours Serology Tests for Histoplasma Capsulatum Detection of specific antibodies ○ Complement Fixation Test ○ Immunodiffusion Test ○ Direct Fluorescent Antibody Test ○ Counterimmunoelectrophoresis Test Closer look at Yeast phase of Histoplasma capsulatum. Laboratory Diagnosis of Histoplasma capsulatum Culture: LYSIS CENTRIFUGATION METHOD Used to enhance the recovery of H. capsulatum 10 ml blood w/ anticoagulants & reagents to lyse RBCs; centrifuge, Pellets with yeasts are inoculated in culture medium Treatment for Histoplasma Capsulatum Acute Pulmonary Histoplasmosis Management: ○ Supportive therapy and rest are the primary approaches. Treatment for Mild to Moderate Infection: ○ Itraconazole is the recommended treatment. Disseminated Disease Treatment: ○ Amphotericin B is the systemic treatment of choice for disseminated histoplasmosis. ○ This treatment is often curative, but patients may require prolonged treatment and monitoring for relapses. @mlstranses | 5 AIDS Patients Treatment Approach: ○ AIDS patients may experience relapses despite curative therapy in other individuals. ○ Maintenance therapy with itraconazole is necessary for AIDS patients to manage and prevent relapses. a. b. Traumatic autoinoculation Contamination of an open wound with infectious material 2. CHRONIC CUTANEOUS BLASTOMYCOSIS Blastomycosis Most common on exposed skin surfaces (face, hands, wrists, lower legs) 3. DISSEMINATED BLASTOMYCOSIS Chronic suppurative and granulomatous infection ○ With pus and granules ○ Common with Dogs and Cats (Zoonotic) inhalation of the conidia, hyphal fragments Lungs; long bones, soft tissue and skin ○ Dissemination may occur to any organ but preferentially to the skin and bones Serology Tests: Complement Fixation, Immunodiffusion, Enzyme Immunoassay Test Causative agent: Blastomyces dermatitidis Biology of Blastomycosis Extrapulmonary sites most common disseminated form: cutaneous type Skin, bones, Genitourinary tract, Central nervous system, and spleen 4. PULMONARY BLASTOMYCOSIS Most common from Pulmonary lesion heals by fibrosis and resorption ○ Scarring is prominent compared to TB Rarely calcifies 1. 2. 3. 4. 5. 1. In the environment (Rainforests), Blastomyces exists as mold with septate aerial hyphae. The hyphae produce spores. These spores are either inhaled, or inoculated into the skin of a susceptible host. The warmer temperature inside the host signals a transformation into a broad-based budding yeast. The yeast may continue to colonize the lungs or disseminate in the bloodstream to other parts of the body, such as the skin, bones and joints, organs, and central nervous system. Clinical Manifestations of Blastomycosis PRIMARY CUTANEOUS BLASTOMYCOSIS Laboratory Diagnosis of Blastomyces dermatitidis MICROSCOPY calcofluor white or KOH prep of pus, exudate, sputum yeast cells are large and have thick walls MYCELIAL PHASE Delicate, septate hyphae Round or pyriform conidia borne signify on conidiophores resembling “lollipops” Has shorter conidiophore than S. boydii ; Described as PEDUNCLE YEAST PHASE Biopsy tissues or body fluids: Hematoxylin & Eosin stain Large-spherical, 8-15 microns (u) Thick-walled yeast A single bud connected to its parent cell by a broad base @mlstranses | 6 ○ Adult type (Pulmonary & Disseminated) affects the lungs, lesions in the mouth and tongue Microscopic Characteristics of Paracoccidioides brasiliensis MYCELIAL PHASE Macroscopic characteristics of Blastomyces dermatitidis Small, sepate, branched hyphae with intercalary (in between septum) & terminal chlamydospores YEAST PHASE Large, round, or oval cells Multiple buds attached to the parent cell by a narrow base “Mariner’s wheel” Chlamydospores can grow at the tip of the hyphae or in between septum 7-21 days Sabouraud Dextrose Agar/Inhibitory Mold Agar ○ White, waxy, yeast-like, becoming cottony with white aerial mycelium; turns tan to brown with age Brain-Heart Infusion Agar with Blood ○ Cream to tan, waxy, wrinkled Serology Tests for Blastomyces dermatitidis Measurement of Complement Fixation Antibodies has not yet been proven reliable Yeast phase provides a more specific Antigen Immunodiffusion test = more sensitive and more specific than Complement Fixation Test Difference between H. capsulatum and P. brasiliensis lies within their forms – mariners wheel appearance of P. brasiliensis is in the yeast phase while for the H. capsulatum its in the mycelial phase Macroscopic Characteristics of Paracoccidioides brasiliensis Treatment for Blastomyces dermatitidis Itraconazole (Oral) and Amphotericin B (Intravenous) Paracoccidioidomycosis Chronic granulomatous infection Begins as a primary pulmonary infection MOT: Inhalation of fungal structures Asymptomatic but may disseminate Infects: Nasal or oral cavity, gingiva, and conjunctiva Do not spread from person-to-person Complement fixation, Immunodiffusion test Clinical Manifestations of Paracoccidioidomycosis Asymptomatic Form (Most of the time) ○ Initial form ○ Latent Symptomatic Form ○ Juvenile type swollen lymph node and skin lesions Very slow-growing Sabouraud Dextrose Agar ○ White, glabrous, leathery colony; tan-brown with age Brain Heart Infusion (BHI) ○ Cream to tan, moist, wrinkled colony which turns waxy with age Serologic Tests for Paracoccidioides brasiliensis infxn Immunodiffusion Test is extremely useful Complement Fixation Test is quantitative and useful for assessing prognosis, but cross-reactions occur with other fungi @mlstranses | 7 APPENDIX: MYCELIAL AND YEAST FORM OF THE FOUR SYSTEMIC MYCOSES MYCELIAL FORM YEAST FORM MYCELIAL FORM YEAST FORM COCCIDIODOMYCOSIS (Coccidioidies immitis) BLASTOMYCOSIS (Blastomyces dermatitidis) HISTOPLASMOSIS (Histoplasma capsulatum) PARACOCCIDIOIDOMYCOSIS (Paracoccidioides brasiliensis) @mlstranses | 8 [MLS 415] Mycology and Virology M2: Opportunistic Mycoses Professor: Joanne Krystine Tago Date: March 8, 2024 TYPES OF OPPORTUNISTIC MYCOSES Candidiasis, systemic Cryptococcosis Aspergillosis Zygomycosis/ Mucormycosis Hyalohyphomycosis Pneumocystosis OPPORTUNISTIC MYCOSES Fungal infections in the body which occur almost exclusively in immunocompromised individuals The causative agents are common and found around us → usually have very low inherent virulence ○ Not asymptomatic; Not self-limiting ○ Usually infected are IMMUNOCOMPROMISED Candidiasis Most frequently encountered Endogenous in origin or nosocomial Fungemia & disseminated infection Candida spp. = normal microbiota in the oropharynx, gastrointestinal tract, genitourinary tract, skin In healthy individuals, Candidiasis are usually due to impaired epithelial barrier Systemic type of Candidiasis are usually seen on patients with cell-mediated immune deficiencies Predisposing Factors - Candidiasis Alteration in the normal skin and mucous membrane barriers (most common for immunocompetent individuals) Prolonged antibiotic administration Use of immunosuppressive drugs Diseases of the immune system Etiologic Agents - Candida spp. Candida albicans (most common) Candida parapsilosis Candida tropicalis Candida glabrata Antigenic Structures - Candida spp. 2 serotypes: A and B Proteases – breakdown of protein structures Enolase – glycolytic enzyme; immunodominant antigen in disseminated infections Heat shock proteins – family or proteins produced by cells usually in response to exposure to stressful conditions (extreme heat, cold or overexposure to UV light) ○ Discovered during wound healing and tissue repair Clinical Manifestations - Candida spp. OROPHARYNGEAL CANDIDIASIS (common) ○ seen on 5% newborns, 10% elderly patients ○ commonly seen on immunocompromised patients with Diabetes mellitus, leukemia, lymphoma, neutropenia, HIV ○ rarely seen healthy adults ○ includes: oral thrush; glossitis (inflammation of the tongue) or stomatitis (inflammation of mouth and lips) ○ the milky-like curd can be found in the buccal mucosa, tongue, gums, and sometimes far back into the pharynx may have a burning sensation, dryness of the mouth, loss of taste, problem in swallowing and drinking Perleche Oral thrush Vulvovaginitis “Diaper rash” in children Eye infections CUTANEOUS CANDIDIASIS ○ Intertrigo ⎼ commonly seen on folds of skin such as the axillary area, groin, submammary folds etc; skin barrier is damaged due to constant exposure to heat and friction @mlstranses | 1 ○ ○ Chronic Candida Onychomycosis – infection usually occur underneath the nails causing it to weaken and produce too much nail debris nail almost separates the nail bed nails are disfigured; crumpled up towards the center and obvious thickening usually seen on: DM, hypothyroidism, Addison's disease, patients with malfunctional thyroid, malnutrition, and malignancies Paronychia ⎼ infection of the fingernails, especially around the cuticle area commonly seen on individuals who constantly have their fingers wet or constant contact with flour (for individuals who work on bakeries) Intertrigo Chronic Candida Onychomycosis [UPPER L to R] PAS and Calcofluor white [LOWER L to R] IF and GMS MACROSCOPIC 24-48 hours (rapid growers) raised, cream-colored, opaque, 1-2 mm medium hyphae may be observed after several days CHROMAGAR CANDIDA (apart from SDA) ○ useful in differentiation Candida spp. as they are allowed to produce different colors ○ C. albicans ⎼ green; C. tropicalis ⎼ blue C. glabrata ⎼ purple; C. krusei ⎼ pink Paronychia Laboratory Diagnosis SPECIMEN skin and nail scrapings urine, sputum, bronchial washing, CSF or pleural fluid, blood Tissue samples (for cutaneous types) DIRECT MICROSCOPIC EXAM 10% KOH, Parker ink or calcofluor white budding yeast cells (blastoconidia) pseudohyphae: strongly Gram (+) CORNMEAL AGAR WITH TWEEN80 (CMA-T80) Identification of Candida spp.a nd other yeasts (pseudohyphae, chlamydospores, blasto-, & arthroconidia) ○ Tween 80: stimulates the conidiation by reducing the surface tension of the culture media DALMAU Expected results: C. albicans ○ with: chlamydospore & blastoconidia arranged along pseudohyphae DALMAU PLATE TECHNIQUE A procedure on planting organisms on either rice meal agar or corn meal agar to allow conidiation Procedure: 1. divide the rice agar plate into sections 2. label the sectors with the number of the tested strain 3. inoculum from the primary media will be inoculated into the rice meal agar 4. after initial streaking, make 4 cuts @mlstranses | 2 C. neoformans - Round to oval Rare, usually not separated by capsule seen Saccharomyces - Large and spherical Rudimentary H sometimes present Numerous, Maybe present but Septated hyphae is resemble difficult to find present Geotrichum Trichosporon Commonly encountered yeast in CMA-T80 Agar 5. 6. 7. Cover with a coverslip the cut portion to introduce a microaerophilic environment Incubate the agar plates aerobically @ 25-30 deg C for 24-48 hours Examine the agar plate under the microscope GERM TUBE hypha-like extension with no constriction at the point of origin ○ look likes it was pulled away from the mother cell formed upon incubation w/ serum at 37°C for 1-3 hours similar to pseudohyphae but it has construction CRYPTOCOCCOSIS An acute, subacute or chronic fungal infection that has several manifestations infections are mild or asymptomatic, or does not require antifungal treatment for immunocompetent individuals Disseminated Disease: → Meningitis In 2/3 of patients. Cryptococcal meningitis could have microscopic lesions on the brain; life-threatening and requires aggressive therapy commonly in patients with AIDS Acquired through inhalation Found in soil Etiologic Agents - Cryptococcus spp. C. neoformans primarily present in soil contaminated with bird dropping, particularly of pigeons C. gattii usually present in soil around trees saprophyte bat, or bird (pigeon) droppings, decaying vegetations, fruit, plants MOT: inhalation (lungs) disseminated (meninges & other sites) endocarditis, hepatitis, renal infection, pleural effusion Attacks IMMUNOCOMPROMISED individuals Organism Arthroconidia Blastoconidia Pseudohyphae or Hyphae C. albicans - Spherical clusters at regular intervals on pseudohyphae Chlamydoconidia on hyphae C. glabrata - Small, spherical, tightly compact None C. krusei - Elongated. clustered at Branched septae of pseudohyphae pseudohyphae C. parapsilosis - Present but not characteristic Sagebrush like. Giant hyphae Organism Arthroconidia C. kefyr (pseudotropicalis) - Elongated, parallel to PH present, not pseudohyphae characteristic C. tropicalis - Randomly appear on PH present, not PH & H characteristic Blastoconidia Laboratory Diagnosis (C. neoformans) DIRECT MICROSCOPY → specimen depends on the infected part spherical, single or multiple budding, thick walled yeast cell (2 to 15 um) wide, refractile polysaccharide capsule CAPSULE ○ India ink preparation of CSF (visualization of the thick cell wall) ○ Mucicarmine ○ Masson-Fontana Pseudohyphae or Hyphae [LEFT] Pleural fluid on KOH [RIGHT] Capsule on India ink @mlstranses | 3 MACROSCOPIC Colonies in 1-5 days (rapid growers) smooth, white to tan, mucoid, gelatin-like colonies (soap-bubble) brown-black colonies on Niger Seed Agar ○ their ability to produce melanin is enhanced vue to the enzyme, phenoloxidase SDA without cycloheximide ○ Cryptococcus is sensitive to cycloheximide membrane filter technique (optimal recovery) ○ for watery/fluid-like specimen Organism FERMENTATION Urease Nitrate Reducti on G M S L C. albicans + + - - - - C. tropicalis + + + - - - C. parasilosis + - - - - - C. glabrata + - - - - - C. neoformans - - - - + - Geotrichum - - - - - - T. beigelii - - - - + - [LEFT] SDA without cycloheximide [RIGHT] Niger Seed Agar Identification of Yeast Cells Latex test for cryptococcal capsular antigen (serologic test) ○ positive in CSF or blood specimens in >90% of patients with meningitis; generally specific ○ false-positive results may occur (usually if rheumatoid factor is also present) partner with RF testing TREATMENT For localized pulmonary disease: fluconazole For meningitis or other severe infection: amphotericin B (with or without flucytosine, followed by fluconazole) Organism Caps ule Germ tube Blastoconidia Arthroconidia Chlamydospore C. albicans - + + - + C. tropicalis - - + - V C. parasilosis - - + - - C. glabrata - - + - - C. neoformans + - + - - Geotrichum - - - + - T. beigelii - - + + - ASPERGILLOSIS Infection, usually of the lungs, caused by Aspergillus spp. Immunocompetent individuals may be asymptomatic Acquired through inhalation Manifestations Invasive lung infection (common) Pulmonary or sinus fungus ball ○ usually develop on the open spaces in the body ○ fungus ball / aspergilloma ⎼ an intact mycelium of aspergillus usually grows in the cavities in the lungs remnants of the damaged cells on the previous infections can also grow in the sinus area, ear canals ○ gradually enlarges and causes destruction of lung tissues especially around it Allergic bronchopulmonary aspergillosis → for individuals with asthma or cystic fibrosis, they develop allergic reaction with coughing, wheezing, and fever if the Aspergillus colonizes the lining of their airway Cerebral aspergillosis Disseminated types: (immunocompromised) ○ Keratitis, otomycosis ○ Onychomycosis ○ Sinusitis, endocarditis, CNS infection @mlstranses | 4 MACROSCOPIC rapid grower (2-6 days) fluffy to granular, white to blue green colonies [LEFT] Aspergilloma on MRI [RIGHT] Cerebral aspergillosis ZYGOMYCOSIS or MUCORMYCOSIS An infection caused by a diverse group of fungal organisms either from the order Mucorales Rhizopus, Rhizomucor, Mucor, Absidia, Cunninghamella Symptoms may frequently result from invasive necrotic lesions in the nose and palate, pain, fever, orbital cellulitis, purulent nasal discharge CNS symptoms may follow Etiologic Agents - Aspergillus spp. Aspergillus fumigatus (representative organism) ○ most common agent isolated in immunocompromised individuals Aspergillus flavus Aspergillus niger ○ commonly isolated from fungus ball and otitis externa Aspergillus terreus ○ difficult to isolate Pulmonary symptoms are usually severe and includes productive coughing, high fever, and dyspnea Decaying vegetable matter, old bread or soil Inhalation Rhinocerebral infection (nasal mucosa, palate, sinuses, brain) Lungs, GIT Less common than Aspergillus Individuals with DM or under immunosuppressive drugs has greater risk of acquiring the infection Pathogenesis Binds to fibrinogen and laminin in the alveolar basement membrane Produces gliotoxin (may inhibit phagocytosis) Neutrophils can adhere and kill the hyphae → but the fungal organism is a rapid grower; hence, they can overwhelm the neutrophils May invade pulmonary and vascular tissue ⇒ thrombosis and necrosis ⇒ hematogenous spread Laboratory Diagnosis: (A. fumigatus) MICROSCOPIC Septate hyphae that usually show dichotomous branching ○ branches usually emerged at 45 deg C from the parent hyphae Dome shaped vesicle with bottle shaped phialides Cutaneous form of Mucormycosis Rhinocerebral infection usually manifestation of Mucormycosis [Upper LEFT] H&E [ Upper RIGHT] Methenamine SIlver Etiologic Agents Generally called as: Zygomycetes ○ Rhizopus (R. oryzae) ○ Mucor ○ Absidia ○ Cunninghamella @mlstranses | 5 Laboratory Diagnosis: (Zygomycetes) MICROSCOPIC tissue specimens or exudates “lollipop-like” fruiting body large ribbon-like branching, non septate hyphae and the presence of zygospore sac-like sporangia connected to the sporangiophore Rhizoids ⎼ hyphal structure that act like roots Stolons ⎼ hyphal structure that act like connectors ○ part of the hyphae will grow away from the main branch, then land on another area to grow another set of fruiting bodies CUNNINGHAMELLA With stolons Pyriform sporangia Funnel shaped vesicle (APOPHYSIS) Erect, straight, branching sporangiophores Globose or pyriform shaped vesicles One-celled, globose to ovoid sporangia C. bertholletiae MACROSCOPIC fluffy, white to gray to brown colonies 24-95 hours (rapid growers) grayish hyphae with brown to black sporangia ○ what is seen on expired bread ORGANISM MUCOR COLONY RHIZOPUS Sporangiophores (stem) Sporangia (bag of spores) Sporangiospores (inside the sporangia) No rhizoids and stolons Unbranched sporangiophores (B) Sporangia (A) Rhizoids appear at the point at which the stolon arises [UPPER L to R] Mucor and Absidia [LOWER L to R] Rhizopus and Cunninghamella ABSIDIA Branched sporangiophores Sporangiophores arise between nodes from which rhizoids are formed @mlstranses | 6 HYALOHYPHOMYCOSIS Fusarium ○ common environmental flora ○ several manifestations Mycotic keratitis (common) − occurs after traumatic corneal implantation ○ other forms: sinusitis, wound (burn) infections, allergic fungal sinusitis, RT secretions Laboratory Diagnosis MICROSCOPIC small septate hyphae large sickle-or boat-shaped macroconidia microconidia are produced (seldom) MACROSCOPIC grow rapidly (2-5 days) fluffy to cottony and may appear pink, purple, yellow, or green ○ F. oxysporum: purple ○ F. solani: pink TALAROMYCES Miscellaneous organism previously called Penicillium molluscum contagiosum-like ○ a cutaneous infection ○ molluscum contagiosum is a viral infection cutaneous ulcers of extremities other forms are bronchopulmonary, endocarditis T. marneffei – clinically significant species Laboratory Diagnosis MICROSCOPIC hyaline & septate hyphae brush-like conidiophores with metulae from which phialides with chains of conidia arise similar to Aspergillus but with minimal conidiation “skeleton fingers” MACROSCOPIC green, blue-green, white, pink colonies (saprophytic) typical yellow-green to pink colony with distinctive red diffusible pigment PNEUMOCYSTOSIS A lung infection Almost exclusively seen in individuals whose immune systems has been compromised by HIV or undergoing chemotherapy ○ for HIV/AIDS patient, pneumocystis is usually a terminal event Etiologic Agent Pneumocystis jiroveci (P. carinii) opportunistic atypical fungus causing pneumonia in immunocompromised hosts specimen: BAL fluid or lung biopsy does not grow artificially CHOLESTEROL in cell membrane Has cystic and trophozoite forms it falls between Ascomycetes and Basidiomycetes was previously identified as a protozoa but due to molecular studies, they are identified as true fungal organism @mlstranses | 7 Laboratory Diagnosis Cannot be cultured, can only be viewed on smears Trophozoite forms ○ pleomorphic ○ evident on GIEMSA Cystic forms ○ 4 to 7 um, does not bud, with intracystic bodies ○ methenamine silver; calcofluor white ; IF stain [LEFT] Methenamine Silver [RIGHT] Immunofluorescence stain Treatment Similar to TB patients Trimethoprim-sulfamethoxazole @mlstranses | 8 [MLS 415] Mycology and Virology M3: Introduction to Virology Professor: Joanne Krystine Tago Date: March 11, 2024 INTRODUCTION TO VIROLOGY VIRUS – means poison or noxious agent in Latin VIRION – refers to an entire virus particle Viral Genome: Based on its organization DIFFERENCES VIRUSES BACTERIA FUNGI Not a living organism, but are active No cell ⇒ acellular External structures may be covered by proteins called capsids Genetic material: DNA or RNA (not both) Do not undergo metabolic processes on their own Remain dormant until they attach to a host cell Prokaryotic Genetic material: arranged at the central portion of the structure (anuclear area) → genome is not encased on a membrane No mitochondria but can still produce ATP Has cell wall that is mostly made up of peptidoglycans Has DNA Can live as either aerobic or anaerobic organism Eukaryotic Either single-celled (yeast) or multi-celled (mold) Has cell wall that is made up of chitin and cellulose Genetic material: DNA Can live as either aerobic or anaerobic organism Reproduction: ○ Yeast: budding ○ Mold: hyphal extension or spore formation Commonly seen as mushrooms → ranges from toxic to edible Saprophytes Positive sense – genome is similar to the host cell’s mRNA ○ readable by ribosomes Negative sense – genome is not similar to host cell’s mRNA ○ not readable by ribosomes ○ can travel with RNA polymerase, which helps them to transform from negative sense genome to positive sense genome allowing their material to be read by the ribosomes DNA Viruses Most are double-stranded Except: → Parvovirus RNA Viruses Most are single-stranded Except: → Reovirus Viral Structure Protein coat or Capsid ○ composed of capsomeres ○ symmetry/ shapes ○ capsomeres can assemble into: Helical (Most common), polyhedral, spherical , complex CLASSIFICATION OF VIRUSES DNA VIRUSES ➔ ➔ ➔ ➔ Poxviridae Herpesviridae Adenoviridae Papovaviridae RNA VIRUSES ➔ ➔ ➔ ➔ ➔ ➔ ➔ Picornaviridae FiloCaliciParamyxoTogaOrthomyxoFlavi- ➔ ➔ ➔ ➔ ➔ ➔ ➔ ReoRhabdoBunyaCoronaAenaAstroRetro @mlstranses | 1 Envelope → lipid bilayer consisting of matrix proteins and glycoproteins ○ places viruses into one of 7 groups depending on the: combination of their nucleic acid strandedness (single or double) sense (positive or negative) KEY FEATURES IN BALTIMORE CLASSIFICATION: DNA Viruses are enveloped Except: Parvovirus Adenovirus Papovavirus (Papillomavirus & Polyomavirus) RNA Viruses are enveloped Except: Calicivirus Reovirus Astrovirus Picornavirus Size Measured by nanometer (nm) ○ 1nm = 0.001μm = 1x10-6 mm Cannot be seen using light microscope E/M; vary from 10 – 300 nm Smallest Largest Parvovirus (22 nm) Picornavirus (28 nm) Poxvirus (225 – 300 nm) Paramyxoviridae (150-300 nm) Filoviridae (80x1000 nm) Baltimore Classification Created by David Baltimore Based on how they generate their mRNA Classification system that is mostly used today All double stranded genome are (+) and (-) sense All other genomes are only (+) sense ○ except: Group V only (-) sense ○ Group II can package both negative and positive strand into their capsid so they may also be (+) and (-) sense at the same time Group I viruses ○ remember that double stranded DNA can make mRNA directly Group II viruses ○ while single stranded DNA requires conversion to double stranded DNA to make mRNA Group III viruses ○ double stranded RNA can be used as a template to make mRNA in a similar way the double-stranded DNA ○ but is uses an RNA-dependent RNA polymerase instead of a DNA-dependent RNA polymerase Group IV viruses ○ single stranded (+) sense RNA is already a mRNA ○ can be used to make proteins immediately ○ alternatively, it can be copied into its (-) sense to produce more (+) sense mRNA still requires an RNA-dependent RNA polymerase Group V viruses ○ function exactly like the post-conversion Group IV viruses ○ can produce the (+) sense mRNA from the (-) sense genomic RNA strand using RNA-dependent RNA polymerase Group VI and VII viruses ○ carry a reverse transcriptase to produce DNA from an RNA strand ○ can make double stranded DNA, which can be used to produce the mRNA Group VII viruses ○ genome is comprised of a circular, gapped double stranded DNA some of its DNA is single stranded some of its genome contains RNA as a primer to generate the require strand of single stranded RNA that will eventually become the template for the reverse transcriptase @mlstranses | 2 # CHARACTERISTICS I dsDNA mRNA is transcribed directly from the DNA template II ssDNA DNA is converted to double-stranded form before RNA is transcribed III dsRNA mRNA is transcribed from the RNA genome IV ssRNA (+) Genome functions as mRNA Common cold (picornavirus) V ssRNA (-) mRNA is transcribed from the RNA genome Rabies (rhabdovirus) VI ssRNA viruses w/ reverse transcriptase Reverse transcriptase makes DNA from the RNA genome; DNA is then incorporated in the host genome; mRNA is transcribed from the incorporated DNA dsDNA viruses w/ reverse transcriptase The viral genome is double-stranded DNA, but viral DNA is replicated through an RNA intermediate; the RNA may serve directly as mRNA or as a template to make mRNA VII MODE OF mRNA PRODUCTION EXAMPLE Herpes simplex (herpesvirus) Canine parvovirus Rotavirus HIV Hepatitis B virus VIRAL REACTION TO CHEMICAL AND PHYSICAL AGENTS ETHER FORMALDEHYDE Distinguishes enveloped viruses from naked viruses Enveloped viruses are generally sensitive Naked viruses are RESISTANT The envelope of most viruses is derived from the host cell’s cytoplasmic membrane ○ except Herpes viruses since they derive their envelope from the host cell’s nuclear membrane The presence of an envelope confers instability rather than protection ○ enveloped viruses are more sensitive than naked viruses, especially to heating, drying, detergents and lipid solvents Destroys viral infectivity by reacting with nucleic acid SS genomes are inactivated more readily than DS genome ○ because single stranded genome has lesser products to work on Minimal adverse effect on antigenicity of proteins ○ Still able to cause infection Some studies showed that the inactivation of formaldehyde has an effect of the early step of viral replication @mlstranses | 3 reduces the ability of the Polio virus to bind to the receptors; thus, the virus will not be able to gain entry into the host cell ○ abolishes the infectivity of the viral DNA; thus, it limits the virus from infecting only few cells Activity of formaldehyde: creates crosslinks; materials far apart will be pulled together ○ RADIATION Ultraviolet, x-ray, high-energy particles inactivate viruses ○ even naked viruses Ionizing and Non-ionizing ○ The lower the wavelength the higher chances that it causes an effect Infectivity is the most radiosensitive property ○ this property is embedded in the genome Exposure to radiation damages part or components of the genome ○ replication may be a problem HEAT Enveloped viruses rapidly drop in titer at 37°C Icosahedral viruses lose infectivity after several hours at 37°C Viral infectivity is destroyed by heating at 50- 60°C for 30 minutes ○ except HBV and Polyoma viruses HBV is a very resistant pathogen ➔ can survive heating at 60°C even up to 4 hours ➔ can be inactivated by high temperatures such as in autoclaving (121°C) or direct heat ○ Polyoma viruses are also inactivated by high temperature ≥70°C is required to affect thermal inactivation VIRAL REPLICATION Viral Replication → term used to indicate the formation of biological viruses during the infection process inside the target host cell Viruses must first penetrate and enter the cell before viral replication can occur Purpose: multiplication and survival ADDITIONAL INFORMATION: (from the video) Viruses are transferred as particles known as virions Once the virions enter the host cell, it disassembles and the viral genome interfere with cellular processes 1st Stage: ATTACHMENT & PENETRATION Virion Attachment: Viruses cannot pass through biological membranes on their own. They utilize membrane proteins to attach to specific receptors on the surface of host cells. Membrane Proteins Involved: These may include ○ sialic acid-rich glycoproteins, ○ proteoglycans (heparan sulfate), ○ receptors ( LDL or CD4) ○ proteins forming tight junctions such as (occludin and claudin) Entry Mechanisms: Enveloped viruses fuse with the host cell membrane after attachment, releasing their capsid into the host cytoplasm. ○ Non-enveloped viruses may be endocytosed upon receptor binding and transported along cytoskeletal filaments in the cytoplasm. ○ 2nd Stage: UNCOATING Capsid Disassembly: The viral genome, often RNA in RNA viruses, needs to enter the host cell cytoplasm. ○ The viral capsid is disassembled, triggered by factors like endosomal pH changes, leading to the release of the viral genome. Genome Release: Once released in the cytoplasm, the viral RNA (if required) may undergo transformations, such as mRNA transcription, initiating viral protein translation. ○ Viral mRNA often contains sufficient information to encode multiple proteins, facilitated by specific folding patterns or internal ribosome entry sites (IRES). 3rd Stage: REPLICATION/ SYNTHESIS Early Protein Synthesis: The first viral proteins synthesized are typically involved in replication processes. These proteins are catalytically active and produced in smaller amounts initially. Late Protein Synthesis: Structural proteins necessary for forming new capsids are subsequently produced in larger quantities. 4th Stage: ASSEMBLY Once sufficient viral components, including RNA replication products and structural proteins, are produced, they self-assemble into new virions. 5th Stage: RELEASE (1) Exocytosis: Some viruses exit the host cell through exocytosis, where viral particles travel through the endoplasmic reticulum and Golgi apparatus before being released. ○ Maturation often occurs in different cell compartments, depending on pH. (2) Budding: Viral proteins are incorporated into the host membrane, forming a new envelope for the virion. @mlstranses | 4 ○ Viral components assemble directly at the budding site, which could be the endoplasmic reticulum, Golgi apparatus, or plasma membrane. (3) Cell Lysis: Many non-enveloped viruses are released via cell lysis, where the virus disrupts the host cell's plasma membrane, leading to cell death. ○ DNA Viruses DNA viruses follow a similar replication process but have some variations: ○ Nuclear Import: The viral DNA needs to be delivered into the host nucleus for transcription. Inside the nucleus, viral DNA may undergo transformations before transcription. ○ Assembly and Release: Viral structural proteins are often transported into the nucleus for assembly. ○ Virions then penetrate the nuclear membrane and are released via vesicle formation. Adsorption (Attachment) Binding between viral capsid proteins and specific receptors on host cell membranes Viral tropism (specificity) Glycoproteins, proteoglycans, receptors (LDL & CD4), occludins & claudins Attachments triggers changes on the viral envelope proteins or viral surface proteins (in the case of naked viruses) ○ results in the fusion of viral and cellular membranes SYNTHESIS PHASE a.k.a. Viral Entry Virions enter the host cell through: ○ membrane fusion Envelope is left at the cell membrane; which combines w/ the cell membrane Causes change in conformation ○ receptor-mediated endocytosis Due to Cytokines For naked viruses Uncoating Physical separation of nucleic acid from its protein coat Mediated by cellular enzymes Initiated by different triggers such as: ○ endosomal pH ○ presence of cellular enzymes The environment gradually becomes acidic which affects the capsid proteins Virus begins to make copies of itself Inhibition of host cell DNA Early: synthesis of nucleic acids catalytically active and would require only a small amount to be synthesized “early protein” Late: synthesis of structural proteins would require large quantities of structural proteins “large proteins” Location of Viral Genome Replication Nucleus Cytoplasm DNA viruses, except Poxvirus ○ poxviruses are very large (300nm) ○ would not fit into the host cell’s nucleus ○ synthesis occur in the cytoplasm Uncoating occurs in the cytoplasm followed by the transportation of the genome into the nucleus RNA viruses, except for retroviruses & Influenza virus ASSEMBLY or MATURATION Penetration causes destabilization of capsid proteins, separating capsomeres from one another and exposing the genome Newly formed nucleic acids are enclosed by capsids Maturation: proper orientation of the structures inside the virus ○ in some viruses, it occurs in place as they are assembled within the cell ○ while other viruses may undergo maturation once they are separated from the host cell RELEASE Two Mechanisms: ○ Rupture or lysis ⎼ for naked viruses ○ Budding or exocytosis ⎼ for enveloped viruses For HIV: → it undergoes maturation after release SPECIMEN COLLECTION FOR VIRAL STUDIES Collect specimens as soon as possible after the onset of symptoms ○ the chance of patient recovery is best during the first 3 days after onset of symptoms ○ patient recovery is reduced the longer the specimen is collected from the patient @mlstranses | 5 Collect autopsy samples ASAP after death before tissues start decomposing All specimens (except feces) must be collected in a designated sterile container and kept cool on ice or refrigerated (not frozen) Specimen Optimal specimen collection schedule Cerebrospinal fluid (CSF) for most PCR or culture samples, they should be held at 4 deg C and must arrive in the laboratory within 24 hours Observe aseptic technique and proper labeling ○ Volume Within 7 days of onset 1-2 mL Amniotic fluid 3 mL Vitreous fluid (eye) 1 mL Urine Specimen handling instructions Within 2 weeks of onset 5 mL Blood 5-7 mL In yellow ACD tube Bone marrow aspirate 2-5 mL In yellow ACD tube Bronchoalveolar lavage (BAL) 3-5 mL Swabs (nasal, throat, nasopharyngeal, vesicle/ lesion, rectal, eye/conjunctiva, genital) NP: within 5 days VESCL/GEN swabs - as early as possible Swab in M4 Aspirates (nasal, tracheal, sinus, vesicle) 1-2 mL Nasal wash Within 5 days of onset Feces Within 2 weeks of onset 3-5 mL 5 mL Tissue Variable Nasopharyngeal Swab or Aspirate In viral transplant media M4 Aspirates are superior to swabs for recovering viruses Procedure is time-consuming and uncomfortable for the patient For COVID-19 specimens, nasopharyngeal swab is the specimen of choice ○ more convenient Best for isolation of respiratory syncytial virus, influenza, parainfluenza, rhinovirus In container without preservatives In sterile saline or viral transport media Bronchial and Bronchoalveolar Washing Excellent specimens for detection of viruses infecting the lower RT Includes tracheal aspirates and sputum Should NOT be placed in a container with the universal transport media ○ should only be placed on a clean or sterile container E.g. Influenza virus, adenoviruses Conjunctival Swab Nasal Wash Aspirate 10 mL (for adult) sterile NSS into a sterile syringe or bulb Tilt head back and apply pressure to one nostril Have patient hold breath and quickly “squirt” 5 mL NSS into open nostril Tilt head forward to allow fluid to drain or expel into sterile collection cup Repeat with other nostril, if possible Transfer this fluid to the sterile conical centrifuge tube Evert the lower eyelid and gently rub the conjunctival surface with a mini-tipped flocked swab 5-10 ml fresh diarrheic stools or 5-10g formed or soft stools Rectal swab may be acceptable ○ Insert a swab 3-5 cm into rectum to obtain feces Rota-, enteric adeno-, enteroviruses (do not grow in cell cultures) Stool and Rectal Swab Throat Swab Inflamed or purulent areas of the posterior pharynx Swabs are allowed to touch the posterior pharynx at the back and the tonsillar fossae Best for isolation of enteroviruses, adenoviruses, herpes simplex viruses Urine Virus recovery may be increased by processing multiple (2-3) specimens Specimen: at least 10 ml of a clean voided, first morning urine CMV, mumps, rubella, measles, polyomavirus, adenoviruses @mlstranses | 6 Vesicular lesions ○ intact, well-defined, and shows characteristic features of the viral infection HSV, Varicella-Zoster virus, Enteroviruses ○ VZV causes Chickenpox and Shingles Usually found in the Dorsal Root Ganglia (clusters of nerve cell bodies located along the spinal cord) These ganglia serve as reservoirs for certain neurotropic viruses; Viral particles can establish latent infections within the DRG, where they remain dormant until reactivation occurs. EDTA is preferred for PCR test Heparinized spx. are not used in PCR Test ○ citrate or heparin may be used if for culture 2 ml specimen is acceptable for pediatric patients Detection is best done by separating and culturing WBCs Polymorphprep (Na metrizoate + dextran) ○ MNs, PMNs are isolated from RBCs in one step ○ Skin and Mucous Membrane Lesions A Tzanck smear is a simple and rapid diagnostic test used to detect viral infections, particularly those caused by HSV and VZV. Bone Marrow Procedure: Scraping cells and fluid from the lesion onto a glass slide. Staining and examining under a microscope for viral cytopathic effects like multinucleated giant cells and inclusion bodies. Interpretation: Presence of multinucleated giant cells and inclusion bodies suggests an active viral infection, It helps differentiate viral infections from other causes of vesicular lesions. Collected by aspiration ○ only done by physicians Sterile tube with EDTA Should be stored and transported at room temperature ○ should be prioritized – bcs BLASTIC cells are sensitive to cold temps Parvovirus B19 – infects children Tissue Collected by biopsy Small pieces of tissue should be placed in Viral Transport Media (VTM) or sterile Phosphate Buffer (PBS) Do NOT add the swab to the transport media Rotaviruses, adenoviruses, caliciviruses, astroviruses, Norwalk virus, and a group of Noroviruses. Serum for Antibody Testing Sterile Body Fluids other than Blood (1) CSF, (2) pericardial, (3) pleural and (4) peritoneal fluids Specimens are collected aseptically by the physician 0.2 mL for PCR test (especially for CSF) 0.5 mL to culture CSF Do NOT dilute in universal transport media Enteroviruses, HSV, influenza virus, CMV Blood Detect CMV ○ occasionally HSV, VZV, enteroviruses, adenoviruses 3-5 ml anticoagulated blood (EDTA, citrate or heparin) collected in a vacutainer tube Acute specimens ○ collected ASAP after the appearance of the symptoms Convalescent serum ○ 2-3 weeks after the acute serum ○ to check the stability of patient’s immune system 3-5 ml serum SPECIMEN TRANSPORT, STORAGE, PROCESSING FOR VIRAL CULTURE Specimens for Viral Culture Do NOT stand at RT or higher Store in ice & transport to the lab ASAP If delay is unavoidable, should be refrigerated @mlstranses | 7 Storage up to 5 days at 4°C ○ 6 or more days at -20°C or preferably at -70°C Before freezing: dilute or emulsify in viral transport medium Transport blood in anticoagulated sterile tube Clotted blood are unacceptable ○ should be processed ASAP ○ if delayed, hold specimen at 2-8°C ○ processing must occur within 12-24 hours ○ serum may be stored for days at 4°C or for weeks / months at -20°C or below before testing Swabbed samples ○ cotton, rayon, dacron (preferred) Must be emulsified in viral transport medium before transporting Cytopathic Effect (CPE) Viral Transport Media Fetal Bovine Serum, albumin, or gelatin Antimicrobials (Gentamicin & Amphotericin B) ○ to inhibit contaminants Gentamicin: bacterial contaminants Amphotericin B: fungal contaminants Examples: ○ Stuart, Amies, Leibovitz-Emory ○ Hanks balanced salt solution (HBSS) ○ Eagle’s tissue culture medium ○ Virocult® and ∑-Virocult® METHODS OF DETECTING VIRUSES Cytology and Histology Electron Microscopy Immunodiagnosis Molecular Detection Cell culture Viral Serology CYTOLOGY AND HISTOLOGY Specimens: tissue samples or body fluids Cytology stains: ○ Papanicolaou (Pap) ○ Giemsa stain ○ H&E detects inclusions & syncytia of CMV, Rabies, HPV, MCV Detects: inclusions: structures that are not supposed to be inside tissues or host cells syncytia: giant cell made up of different cells infected by viruses (HSV or chickenpox infections) Can identify: → HSV, Varicella-Zoster inclusion bodies Cellular changes ○ cell death or lysis ○ syncytia formation ○ inclusion body formation ○ transformation of host cells Morphologic changes in the virus-infected host cells Observable on light microscopy Cell Death or Lysis → Human corneal epithelial cells infected with HSV1 ○ Image A (0 hour) exhibits cobblestone appearance with 90% confluence confluence: refer to the spaces in between cells ○ Image B (8 hours) cytopathic effect could be seen with 80-75 confluence ○ Image C (12 hours) – more cells are gone ○ Image D (24 hours) – 50-60% confluence Cellular Changes: Inclusions or Syncytia Negri body inclusion ○ Rabies Giant cell with “owl’s eye” inclusion ○ CMV @mlstranses | 8 Guarnieri bodies ○ Smallpox Warthin-Finkeldey syncytia formation ○ Measles ○ giant syncria made up of about 40% of infected cells Koilocytes ○ HPV ○ squamous cells with vacuolation “halo” around the nucleus ○ Cervical smears – cytoplasm should be clear Cowdry type A ○ Herpes simplex ○ Very large inclusion Henderson-Patterson or Molluscum body ○ Molluscum contagiosum ○ seen inside blister formations ELECTRON MICROSCOPY When using electron microscope: ○ the material to be viewed should be treated with electron dense material like sodium tungstate provides a dark background ○ then add the electron condensed material ○ the material will be mounted with copper grids Transmission Electron Microscope ○ allows visualization of the internal structures of the virus Scanning Electron Microscope ○ allows visualization of the external structure of the virus [LEFT] TEM: Rotaviruses notice the empty virions (inactivated viruses) [RIGHT] SEM: Adenoviruses notice the icosahedral formation TEM & SEM Useful for detecting viruses that do not grow readily in cell cultures Examples ○ gastroenteritis (Rotavirus, Norwalk agent, Adenovirus) ○ encephalitis (Herpes simplex, Measles, JCV) Immune Electron Microscopy ○ specific antisera is added so viral particles will be agglutinated allowing them to be viewed as groups ○ rapid and accurate morphological diagnosis of viral agents ○ Classical Phase simple add the anti-sera before viewing it under the electron microscope ○ Solid Phase anti-sera is loaded onto the copper grids VIRAL SEROLOGY Indirect evidence of viral infection To evaluate immune status ○ recall acute specimen and convalescence serum To diagnose viral infections if virus cannot be cultivated 1-2 weeks: IgM begin to appear followed a few days later by IgG ○ IgM levels peak in 3-6 weeks and drop to undetectable levels in 2-3 months ○ IgG levels peak in 4-12 weeks and remain for several months, some for life Diagnosis of Active Infection Detection of virus-specific IgM in acute phase serum sample Detection of a four-fold rise in antibody titer between acute and convalescent sera Detection of Viral Antigens Immunofluorescence Assay ○ a microscopic method that detects and visualizes viral proteins expressed in cells via Ag-Ab reactions ○ used for research rather than diagnostic purposes @mlstranses | 9 Enzyme Immunoassay ○ ELISA uses an enzyme to detect the binding of antigen or antibody such as, (1)horseradish peroxidase, (2)alkaline phosphatase, (3)lactoperoxidase, (4)beta galactosidase Principles involved in ELISA are: Antigen-antibody reaction − presence of antigen or antibody is detected in the sample Enzymatic chemical reaction − rate of formation of antigen-antibody complex is used is used to determine the quantity of either the antigen or antibody involved in the reaction – the enzyme catalyzes the colorless substrate to produce a colored product Signal detection and quantification − the intensity of colored product generated by the enzyme and the substrate is detected and measured KEY FEATURES OF ELISA (From the video) Direct ELISA: Involves testing for the presence of an antigen. A known antibody is immobilized or absorbed onto the surface of a microtiter plate well. After rinsing to remove unbound antibodies, the sample suspected of containing the antigen is added. An enzyme-linked antibody specific to the antigen is introduced. ○ If the antigen is present in the sample, it binds to the immobilized antibody and subsequently to the enzyme-linked antibody. A colorless substrate for the enzyme is added. Development of color indicates the presence of the antigen in the sample. Indirect ELISA: Detects the presence of antibodies in a sample. The antigen to be detected is immobilized onto the surface of a microtiter plate well. After washing away unbound antigens, the serum suspected of containing the antibodies is added. An enzyme-linked antibody, capable of reacting with the constant region of other antibodies (i.e., secondary antibody), is then added. ○ If antibodies specific to the antigen are present in the serum, they bind to the immobilized antigen and subsequently to the enzyme-linked secondary antibody. A colorless substrate for the enzyme is added. Development of color indicates the presence of antibodies that recognize the antigen in the sample. Western Blot ○ also called Protein Immunoblotting ○ considered the gold standard test for HIV infection confirmation ○ requires technical expertise; time-consuming NO longer recommended by WHO, CDC, APHC due to: Prone to False (+) Indeterminate results (if infection is early) Long TAT ○ a widely accepted analytical technique used to detect specific proteins in the given sample ○ uses Sodium Dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in the given sample ○ the separated proteins are then transferred or blotted onto a matrix where they are stained with antibodies (used as a probe) specific to the target protein ○ by analyzing location and intensity of the specific reaction, expression details of the target proteins in the given cells or tissue homogenate could be obtained KEY FEATURES OF WESTERN BLOT (From the video) The process described is the Western blotting technique, a powerful tool used to detect and identify specific proteins within a complex mixture. Here's a comprehensive breakdown of the steps involved: (1) Denaturation and Electrophoresis: Proteins within the sample are denatured and coated with sodium dodecyl sulfate (SDS), which imparts a negative charge to the proteins, making them proportional in charge to their mass. The denatured proteins are then separated based on their molecular weight by electrophoresis, where they migrate through a polyacrylamide gel towards the positively charged anode. ○ This separation allows for the visualization of individual protein bands. @mlstranses | 10 (2) Transfer onto a Membrane: After electrophoresis, the separated proteins are transferred from the gel onto a membrane, typically made of materials like nitrocellulose or PVDF (polyvinylidene difluoride). ○ This transfer process, known as "blotting," preserves the spatial arrangement of the separated proteins. (3) Blocking: The membrane is incubated in a blocking solution, often containing milk or bovine serum albumin (BSA). ○ This step prevents nonspecific binding of antibodies and reduces background noise. (4) Primary Antibody Incubation: The membrane is then incubated with a primary antibody specific to the target protein. ○ The primary antibody recognizes and binds to a specific epitope (a unique region) on the target protein. (5) 1st Washing: Unbound primary antibodies are removed by washing the membrane, reducing nonspecific binding and background noise. (6) Secondary Antibody Incubation: The membrane is incubated with a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase. ○ This secondary antibody specifically recognizes and binds to the primary antibody. (7) 2nd Washing: Excess unbound secondary antibodies are removed by washing the membrane, further reducing background noise. (8) Detection: The presence of the target protein is visualized through enzymatic activity. The membrane is exposed to a substrate solution containing a chromogenic or chemiluminescent substrate specific to the enzyme conjugated to the secondary antibody (e.g., HRP). In the presence of the substrate, the enzyme catalyzes a reaction that generates a detectable signal, typically a colored precipitate or luminescent signal, directly proportional to the amount of target protein bound by the primary antibody. MOLECULAR DETECTION Nucleic acid amplification tests such as the PCR are widely used nowadays in identification of viral agents Polymerase Chain Reaction – process done to amplify genetic materials; takes hours, weeks or months ○ a technique that is widely used in molecular biology and genetics that permits the analysis of any sequence of DNA or RNA ○ ○ allows a specifically targeted DNA sequence to be copied and/or modified in predetermined ways this reaction has the potential to amplify one DNA molecule to become over 1 billion molecules in less than 2 hours KEY FEATURES OF PCR (From the video) Polymerase Chain Reaction (PCR) is a widely used molecular biology technique for amplifying specific segments of DNA. It relies on specific oligonucleotide primers, nucleotides for DNA polymerization, and a heat-stable DNA polymerase, typically Taq polymerase from the thermophile Thermus aquaticus. Procedure: 1. Denaturation: The reaction mixture is heated to around 95°C for 30 seconds. a. This step causes the separation (denaturation) of the DNA strands, breaking the hydrogen bonds between them. 2. Annealing: The temperature is then lowered to around 55°C for 30 seconds. a. During this step, the specific oligonucleotide primers anneal or bind to the complementary sequences flanking the target DNA region. 3. Extension: The temperature is raised to around 72°C, which is the optimal temperature for Taq polymerase activity. a. Taq polymerase utilizes the primers as starting points for DNA synthesis, adding nucleotides one at a time to extend the primers and create complementary strands of DNA. 4. Cycle Repeated: The denaturation, annealing, and extension steps constitute one cycle. a. This cycle is typically repeated multiple times, usually around 25 to 30 cycles, to amplify the target DNA exponentially. b. After two cycles, four double-stranded DNA sequences are produced from the original target DNA. 5. Thermocycler: To automate the precise control of temperature changes required for each step of the PCR process, a specialized instrument called a thermocycler is used. a. The thermocycler rapidly cycles through different temperatures, ensuring optimal conditions for denaturation, annealing, and extension. @mlstranses | 11 CELL CULTURE Viruses can be grown in vivo or in vitro IN VIVO: important for the identification and diagnosis for pathogenic viruses in clinical specimens, production of vaccines, and basic research studies host sources serves as an incubator for viral replication (e.g. developing embryo or whole animal) the location within the embryo or the host animal is very important ○ tropism: attacks specific host cells which contains receptors sites for the viruses (e.g. amniotic cavity, chorioallantoic membrane, yolk) IN VITRO: includes bacteriophage grown in the presence of a dense layer of bacteria culture media usually used: 0.7% soft agar for lytic bacteriophages, lysis of bacterial host can be readily observed when there is plaque ○ clear zones where bacterial cells have already lysed Identification of viruses detected on cell cultures are based on: ○ the cell type that support viral replication ○ time of detection of CPE ○ morphology of CPE Conventional Cell Culture → cells grow into a layer on bottom of cell culture flasks or dish ○ cells are moistened and nourished by immersing in cell culture media Dulbecco’s Modified Eagle Medium (comes with bovine serum) ○ incubated for 48 hours at 37°C with 5% CO2 ○ once it has been subcultured in vitro, it becomes a cell line once cells are close together, mitosis is triggered to stop which would results to apoptosis hence, 100% confluence should be avoided by doing subculture Examples: Primary monkey kidney cell (PMK) Human embryonic kidney (HEK) ○ Low-Passage (Diploid) Cell Lines → cell cultures that remain virus-sensitive through 20-50 passages ○ examples: Human diploid fibroblast cells (HDF) derived from human kidney and lung fibroblasts WI-38 and MRC-5 (human embryonic lung) – MRC (Microbiology Research Council) is more common: line 5 or 7 Continuous Cell Lines → cells can be passed and remain sensitive to virus infection indefinitely ○ “immortal cell lines” ○ are usually collected from cancer cells ○ examples: Hep-2 cells HeLa cells − originally cultivated from tumor cells obtained from Henrietta Lacks, who died due to cervical cancer due to HPV (1951) − first continuous cell lines produced Kinds of Cell Culture A cell culture becomes a cell line once it has been passed or subcultured Primary Cell Lines – freshly prepared from cells or tissues ○ extracted by: mechanical scrapings mincing (to release the cells) enzymatic method using trypsin (inhibits cells from sticking together) ○ those that have been passed only once or twice since harvesting ○ further passage results in decreased susceptibility to viral infection ○ usually requires a liquid culture medium in a petri dish or tissue/cell culture flask for cells to have a solid surface for attachment of growth ○ has limited lifespan due to contact inhibition Uses of Cell Cultures Virus Quantification using Plaque Assay – a standard technique used to determine the concentration of infectious viruses in a sample. Objective: The primary goal of the Plaque Assay is to count the number of viruses present in a specific volume of a sample, typically measured in terms of infectious dose. @mlstranses | 12 Procedure: 1. Preparation of Cell Culture: A confluent monolayer of host cells, often referred to as "hot cells," is prepared in a petri dish or a cell culture plate. a. These cells are susceptible to the virus being tested. 2. Virus Infection: The host cell monolayer is then infected with the virus at varying dilutions. a. This is done to ensure that the resulting plaques are countable and not too numerous or sparse. 3. Overlay with Semi-Solid Medium: After infection, the cell monolayer is covered with a semi-solid medium such as agar or carboxymethyl cellulose. a. This medium prevents the spread of the virus to neighboring cells, ensuring that each virus infects only a single cell. 4. Plaque Formation: As the infected cells replicate and lyse, they release viral particles that can infect neighboring cells. a. This results in the formation of visible plaques, which are areas of cell death in the monolayer. 5. Plaque Visualization and Counting: The plaques can be visualized either by (1) direct observation or using an (2) optical microscope. a. Plaque formation typically takes 3-14 days, depending on the virus being tested. b. The plaques are manually counted, and the results are used to calculate the number of plaque-forming units (PFU) per sample unit volume. c. Each plaque is assumed to represent one infectious virus particle. Viral Adsorption: Discard medium from each well and wash cells with PBS. Add 500μl of diluted viruses into each well. Incubate viruses and cells at 37°C for 1 hour. Shake the plates every 15 minutes to ensure uniform viral adsorption. Overlay Infected Cells with Agarose: Prepare an agarose solution containing 0.3% agarose and culture medium, with 2% FBS. Autoclave a 3% agarose solution and culture medium, pre-warmed at 45°C. Mix 4mL of 3% agarose solution with 36mL of culture medium, keep at 42°C. Discard medium from each well and wash cells with 500μl PBS. Add 3mL of 0.3% agarose solution into each well. Keep plates at room temperature until agarose overlay solidifies. Incubate the plates at 37°C for 60-84 hours to allow viral plaques to form. Cell Fixation and Staining: Fix cells with a 3.6% formaldehyde solution for 1-24 hours. Remove formaldehyde solution and wash out agarose overlay. Stain cells with 0.5% crystal violet at room temperature for 1 minute. Rinse each well with tap water. Count and analyze viral plaques to determine viral concentration. Fetal Bovine Serum (FBS): FBS is NOT USED as a cell culture medium in the plaque assay due to its interference with virus growth. ○ However, it is sometimes used as an overlay to protect medical technologists from potential infection. Buffer and Staining: Phosphate buffer solution (PBS) is used for washing the cells, formaldehyde for fixation, and crystal violet for staining. KEY PROCEDURE FOR CELL CULTURE (From video) Cell Seeding: Seed cells into a 6-well plate and incubate at 37°C for 24 hours. Ensure cells reach a confluency of 90-100%. Virus Dilution: Soak the virus at 37°C in a water bath. Use culture media without FBS to dilute the virus. Each tube contains 1080μl of culture medium without FBS. Add 120μl of virus into the first 2 tubes, mix well. Take 120μl of virus into the next tube and repeat to create 10x serial dilutions. Cytopathic Effect → not just for tissues that has been stained in the histopathology laboratory @mlstranses | 13 [LEFT to RIGHT] RSV and HSV Measles Hemadsorption of RBCs & syncytial formation by mumps virus onto cell sheet surface Quantification of Cell Culture CPE Quantitation Negative Interpretation Uninfected monolayer Equivocal (-/+) Atypical alteration of monolayer involving few cells → might be a need to repeat the steps 1+ 1% - 25% monolayer exhibit CPE 2+ 25%-50% monolayer exhibit CPE 3+ 50%-75% monolayer exhibit CPE 4+ 75%-100% monolayer exhibit CPE @mlstranses | 14 BSMLS 3F MYCOLOGY & VIROLOGY 2022 - 2023 MLS 415 | LECTURE | FINALS LESSON 10: DNA VIRUSES PART 1 VIRAL STRUCTURE - OVERVIEW NAKED VS ENVELOPED VIRUSES            Nucleic Acid – either RNA or DNA (never both) Capsid – protects the nucleic acid Envelope – outer layer DNA – double-stranded RNA – single-stranded VIRUSES: NUCLEIC ACIDS Nucleic acid – viruses may contain either DNA or RNA but NEVER both All DNA viruses are double-stranded except Parvovirus DNA VIRUSES DNA Viruses (HHAPPPP) o Hepadnaviridae o Herpesviridae o Adenoviradae o Papillomaviridae o Polyomaviridae o Poxviridae o Parvovirus Note: Papillomaviridae and Polyomaviridae are formerly under Papoviridae CAPSID 3 shapes: o Helical Medically important viruses o Icosahedral o Complex – present in bacteriophages (infects bacteria) (e.g., Poxviridae) Note: Majority of the DNA viruses have icosahedral capsid                 DNA viruses are enveloped except Parvovirus, Polyomavirus, Papilloma, Adenovirus o PaPPA is naked QUIZ TIME Hepadnavirus – enveloped double-stranded DNA virus Herpesvirus – enveloped double-stranded DNA virus Adenovirus – naked double-stranded DNA virus Papillomavirus – naked double-stranded DNA virus Parvovirus – naked single-stranded DNA virus Poxvirus – enveloped double-stranded DNA virus VIRUSES: SIZE Unit of measurement: Nanometer (nm) Vary from 10 – 300 nm Smallest o Parvovirus (22 nm) o Picornavirus (28 nm) Largest – Poxvirus (225300 nm) PARVOVIRUS Naked single-stranded DNA virus Icosahedral capsid 18-26 nm (smallest virus) Transmission – Close contact, probably respiratory droplet Parvovirus B19 o Tropism for erythroid progenitor cells (P antigen)  It will infect the precursors of RBCs since P antigens are expressed on its surfaces  INTERN CUTIEE