Myco-viro-compre.pdf

MYCOLOGY AND VIROLOGY COMPREHENSIVE REVIEWER Kharam B. Molbog, 2019 University of Santo Tomas Medical Technology

INTRODUCTION TO MYCOLOGY MYCOLOGY • Study of fungi • Gk. “mykes”, meaning mushroom • Other related terms: ▪ Puffballs, molds, mushroom, bracket fungi, mildew Importance of Fungi A. Food source (nutrition) B. Food production (Saccharomyces cerevisiae) ▪ Bread making ▪ Beer ▪ Citric acid C. Antibiotic source ▪ Penicillium chrysogenum (notatum) D. Symbiosis ▪ Mycorrhizae = mutualistic relationship between fungi and plant roots o Aids in the absorption of water and minerals from the soil ▪ More than 100,000 species o Only a few are pathogenic (~200); mostly affects the immunocompromised o Most are saprobes or saprotrophs (derive nutrition from decaying matter) Characteristics • Thallophytes ▪ Filamentous appendages ▪ No specialized tissues typical of plants (stem, leaves, conducting tissue) • Eukaryotic (with true nucleus and membrane-bound organelles) • Heterotrophic ▪ Carbon source and source of nutrition = organic compounds • Plant-like, but lack stems and roots and does not possess chlorophyll • Cell walls are made up of chitin (affinity with stains); plants have cellulose

Fungi

Bacteria

Type of Cell

Eukaryotic

Prokaryotic

Cell Membrane

Ergosterols are present

No sterols except in Mycoplasma and Ureaplasma

Cell Wall

Complex carbohydrates (mannans, glucans, chitin)

Peptidoglycan (NAG and NAM)

Spores

Sexual and asexual (for reproduction)

Endospores (not for reproduction); some asexual spores (Actinomycetes)

Metabolism

Heterotrophic/chemoheterotrophic

Heterotropgic, photoautotrophic, chemoautotrophic

Oxygen Requirement

Molds: aerobic Yeasts: facultative anaerobes

Aerobic, facultative anaerobe, anaerobe

pH requirement

pH 5 (acidic)

Neutral pH

Temperature requirement

Molds = RT Yeasts = BT

Depends on the identity of the bacteria

Antibiotic sensitivity

Polyenes, imidazoles, griseofulvin

Penicillins, tetracyclines, aminoglycosides

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Obtain nutrients by means of absorption With membrane-bound organelles ▪ Mitochondria, ER, microbodies, fungal cytoskeleton, microtubules organizing center = growth Inclusions ▪ Vacuoles (lipids) ▪ Glycogen ▪ Spitzenkorper = multi-vesicular bodies; organizing center for growth and morphogenesis (supports directional growth of hypha) Some are encapsulated ▪ Cryptococcus neoformans = polysaccharide capsule; fungal meningitis; common in AIDS patients Live as heterotrophs, saprotrophs, or as parasites More resistant to osmotic pressure (withstand high salt and sugar concentrations) May be NF of mouth and intestinal tract (e.g., mouth = Candida albicans)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

YEASTS VERSUS MOLDS

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Yeast

Mold

Unicellular esp. In liquid media; round; nonfilamentous (no hyphae) Can produce pseudohyphae

Multicellular; filamentous; form hyphae

Reproduce by: budding OR binary fission

Reproduce by fragmentation and spore formation

Grows at body temperature (35-37 oC) Facultative anaerobe

Grows at room temperature Aerobic

Moist, smooth, creamy, or buttery colonies with an alcohol-like odor No aerial hypae Identified using biochemical tests

Dry, cottony with a velvety surface Identified by the appearance of spores and hypha Prefer growing on solid media

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Some fungi exhibit dimorphism (2 forms; can either be a yeast or a mold depending on the temperature) ▪ Mold form = recovered in the laboratory ▪ Yeast form = recovered from body specimens ▪ Most pathogenic fungi are dimorphic Yeasts ▪ ▪ ▪

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Tend to undergo budding Non-filamentous (no filamentous structures) Form pseudophypae (cells that fail to detach from the mother cell give a sausage-link appearance with constrictions; true hyphae have no constrictions) o E.g., Candida albicans Reproduce by budding (e.g., Saccharomyces): asymmetric o Asexual reproduction o Extrusion or maturation of the bud to an independent blastoconidium (daughter cell) o Involves lysis of the yeast cell wall so a blastoconidium can form o As blastoconidium enlarges, the nucleus of the parent cell undergoes mitosis, and the new nucleus is passed onto the daughter cell; a septum forms and the new cell breaks free Reproduce by fission (e.g., Schizosaccharomyces): symmetric o Two cells of equal size are formed Facultative anaerobes Grow best at body temperature

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Molds ▪ ▪

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Moist, creamy, and buttery colonies with an alcohol-like odor due to fermentation Identified by: a. Biochemical testing b. Molecular methods

Multicellular; filamentous Fuzzy or woolly appearance due to the formation of mycelia o Mycelium = mass/aggregate of hyphae a. Thallus (vegetative mycelia) = attachment; embedded in the culture medium; absorption of nutrients b. Aerial = visible; extend above the surface of the colony; responsible for the fuzzy appearance c. Reproductive = supported by the aerial mycelia; produce conidia; fruiting bodies with spores inside Hypha = basic structural unit of a mold Reproduction: fragmentation and spore formation o Fragmentation = breaking apart of fertile hyphae; produce spores o Arthroconidia (arthrospore) = spores formed from fragmentation Colonies: velvety, cottony, dry Classification of hyphae a. According to presence/absence of septations/crosswalls (divisions on hyphae) 1. Septate = frequent cross-walls o Mononucleated (1 nucleus per division) o Most fungi have septate hyphae 2. Pauciseptate (sparsely septate) or coenocytic o Few cross-walls at irregular intervals o Zygomycetes (Rhizopus, Mucor) were once considered as aseptate, but microscopic examination revealed occasional septations b. According to pigmentation 1. Phaeoid (dematiaceous) o Darkly pigmented due to the presence of melanin in the cell wall (melanin = protection against UV light) o Irregular branching structures K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Infections are called phaeohypomycosis or chromoblastomycosis o Examples: i. Altenaria ii. Bipolaris iii. Exophiala iv. Wangiella v. Fonseceae vi. Phialophora vii. Cladosporium Hyaline (moniliaceous) o No/light pigmentation o Infections are called hyalohypomycosis o Examples: i. Acremonium ii. Aspergillus iii. Fusarium iv. Penicillium v. Rhizopus vi. Scopulariopsis o

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According to microscopic appearance 1. Racquet hypha = enlarged, club-shaped areas; resemble tennis racquet; Epidermophyton floccosum 2. Spiral = corkscrew-like and tightly coiled; Trichophyton mentagrophytes 3. Nodular = enlarged knots of closely twisted hyphae; Microsporum canis 4. Favic chandelier = antler-like appearance; Trichophyton schoenleinii 5. Pectinate bodies = look like the teeth of a comb; Microsporum audouinii 6. Rhizoids = root-like processes found in Rhizopus

DIMORPHISM AND POLYMORPHISM • Dimorphism = ability of some fungi to exist in two forms, depending on growth conditions ▪ Mold phase and a yeast (spherule) phase

Yeast = tissue state; seen in vivo; seen in vitro when the organism is growth at 37o C ▪ Mold phase = when grown at RT (22o-25o C) ▪ Species (asexual names): a. Blastomyces dermatitidis b. Coccidioides immitis c. Histoplasma capsulatum d. Paracoccidioides brasiliensis e. Sporothrix schenckii f. Penicillium marneffei Polymorphic fungi ▪ Yeast and mold forms in the same culture ▪ Exophiala spp. ▪

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LIFE CYCLE A. Somatic Phase = feeding or trophic phase ▪ Through the production of extracellular enzymes used to digest nutrients in the substratum and for nutrient absorption B. Reproductive Phase ▪ Asexual ▪ Sexual

REPRODUCTION Sexual Reproduction • Gives rise to the teleomorph or telemorph (perfect state) • Joining of 2 compatible nuclei, followed by meiosis Asexual Reproduction • Gives rise to the anamorph (imperfect state) • Synanomorphs = if a single fungus is able to produce multiple distinct anamorphs • No meiosis; mitosis only • Produces genetically identical progeny • Spores are formed by the hyphae of one organism

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Holomorph = the whole fungus; sexual + asexual phases Fungi Imperfecti (Deuteromycota) ▪ Fungi with no sexual stage (e.g., Candida; Torulopsis, Epidermophyton) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

ASEXUAL REPRODUCTION ▪ •

Mechanisms a. Vegetative reproduction = e.g., fragmentation, budding, fission b. Aerial reproduction = e.g., spore production from fruiting bodies

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Fragmentation ▪ Breaking apart of fertile hyphal elements ▪ Result in the formation of arthroconidia ▪ Coccidioides immitis and Geotrichum B. Budding ▪ Blastoconidia formation ▪ C. albicans, Cryptococcus C. Fission ▪ Symmetric ▪ Schizosaccharomyces D. Spore Formation ▪ Carried out by fruiting bodies (conidiogenous cells = cells from which conidia originate) o Examples of conidiogenous cells: Phialides and Annellides a. P h i a l i d e s = v a s e l i k e s t r u c t u r e s ; f o r m phialoconidia b. Annellides = ringed structures that produce annelloconidia o Production of conidia ▪

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Carried out by sporangium o Production of sporangiospores = develop inside sporangium; arises from a closed fruiting body o Rhizopus Spore formation (conidiogenesis) can occur in 2 forms: A. Blastic ⬥ Protoplasm of the conidiogenous cell is blown out or blasted to form a conidium ⬥ Seen in Candida B. Thallic ⬥ No development of conidium until a septum is formed between the conidium and the parent cell; the conidium originates from the whole of the parent cell ⬥ Seen in Dermatophytes and Coccidioides

Blastic forms o Holoblastic - the parent cell wall layers are involved in blastic daughter cell development; whole parent cell is involved a. Blastoconidia - simplest form and produced by budding (e.g., pseudohyphae forms as in cases of Candida) b. Poroconidia - formed by the daughter cell by pushing through a minute pore in the patent cell o Enteroblastic - the outer cell wall does not participate in the process; only inner cell wall is involved a. Phialoconidia - conidia emerges from a phialide (as seen in Aspergillus, Phialophora, and Penicillium); phialides are flask-shaped ▪ Metulae (sing. metula) - sterile structures that hold up the phialide; cannot form spores ▪ Vesicle - extended portion of the conidiophore; holds the phialides b. Annelloconidia - confida formed from an annelide (as the conidia are released, a distinct ring of cellular material is left, leaving behind a distinct saw-toothed scar/ring appearance at the side of parent cell); e.g., Scopulariopsis, Exophiala • Conidium - produced in a manner that does not involve cleavage; produced singly or in long chains or clusters by conidiophores

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Conidia can be born in: a. Singly b. Long chains (catenulate) o Acropetal - chain formation in which the new (youngest cell) is in the tip; Penicillium (paint brush-like) o Basipetal sporulation - new conidia is at the base while the old conidia is pushed to the tip; Paecilomyces c. In clusters

Thallic forms o Holothallic - the whole parent cell is involved in daughter cell development a. Macroconidia - large, septate, oval-shaped, spindle-shaped, or club-shaped; may be thick- or thin-walled, spiny (echinulate), or smooth wall surface; may appear as a dictyospore/muriform; can be an aleurispore (structure breaks when it is released); multicellular ▪ Cylindrical - Trichophyton ▪ Fusiform - Microsporum ▪ Club-shaped - Epidermophyton ▪ Tu b e rc u l a t e - w i t h s m a l l protrusions ▪ D i c t y o s p o re o r m u r i f o r m macroconidia - mosaic appearance due to horizontal and vertical septa b. Microconidia - smaller, unicellular; borne on the sides of the hypha c. Chlamydoconidia - thick-walled; resistant resting s p o re s ; p ro d u c e d b y ro u n d i n g u p a n d enlargement of the terminal hyphen cells; formed during unfavorable conditions ▪ Terminal - at the tip ▪ Intercalary - in between hyphen elements ▪ Sessile - at the sides o Arthric - daughter cell fragments within the hyphen strand before dispersion

a. Arthroconidia - rectangular/barrel-shaped conidia; derived from the fragmentation of the mycelium at the septum ▪ Presence of dysjunctor cells giving a checkered appearance ▪ Coccidioides immitis and Geotrichum •

Sporangiospores - asexual spores of Zygomycetes; borne in a sporangium ▪ Sporangium - sac-like structures from which sprangiospores arise o Collarette - protrudes from the base of the columella o Columella - extended enlarged tip of sporangiophore o Sporangiophore - the stem o Stolon - connects one sporangiophore to another

SEXUAL REPRODUCTION • Fusion of nuclei of two opposing matching strains; exchange of genetic material • Three distinct phases: 1. Plasmogamy - haploid donor cell (+) penetrates the cytoplasm of recipient cell (-) 2. Karyogamy - fusion of two haploid nuclei to form a zygote 3. Meiosis - gives rise to haploid nucleus (sexual spores

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Sexual spores K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

A. Ascospores - spores enclosed in an ascus following karyogamy (around 8 ascospores enclosed in 1 ascus) o Ascocarp - fruiting body o Paraphyses - hyphal elements in between asci; packing hypha o Apical pore - exit point of ascospores o Types of ascocarps: a. Apothecium - cup-shaped ascu are produced inside a cup b. Cleistothecium - ascocarp is enclosed (no opening) c. Gymnothecium - similar to cleistothecium except the outer wall of the ascocarp is loosely organized; ascu are released through the wall openings d. Ascostroma - asci are produced in locules (cavities) in hard masses of supporting hypha called stroma e. Perithecium - flask-shaped with an opening where ascospores are released

C. Zygospores - thick-walled spores formed by the fusion of 2 hyphal strands (homothallic - same/identical hyphal element) D. Oospores - fusion of cells from 2 separate non-identical hypha

TAXONOMY OF FUNGI • Most causative agents of infections are found in four groups of fungi A. Order Mucorales (Phylum Glomeromycota [Zygomycetes]) B. Phylum Ascomycota C. Phylum Basidiomycota D. Deuteromycota (Fungi Imperfecti) Group

Zygomycota

Ascomycota

Basidiomycota

Deuteromycota

A,gymnothecium; B, cleistothecium; C, apothecium; D, perithecium ; E, ascostroma B. Basidiospores - spores are formed inside a basidium (club-shaped reproductive structure)

Characteristics Rapidly growing; usually found in soil Mycelia: profuse, gray-white; aseptate or sparsely septet Asexual reproduction: sporangiospore Sexual reproduction: zygospores Lid-lifters (lift cover of the petri dish) Contaminates specimens Production of sexual spores called ascospores Asexual reproduction: conidia (arthroconidia, blastoconidia) Molds have septate hyphae Sexual reproduction results in four progeny of basidiospores supported by a club-shaped basidium Hyphae have complex septa Imperfect Fungi Most causes of mycoses belong to this group

Example

Rhizopus Absidia (Lichtheimia) Mucor Rhizomucor

Aspergillus Pseudallescheria boydii Trichophyton Penicillium Cryptococcus neoformans (Filobasidiella neoformans)

Coccidioides immitis Paracoccidioides brasiliensis Candida albicans

PATHOGENESIS • Determinants of pathogenicity: ▪ Thermotolerance ▪ Adaptation to a parasitic lifestyle ▪ Adhesins ▪ Use of enzymes to attach host tissues ▪ Dimorphism K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Evasion of immune response Presence of cell wall molecules that are barriers that resist lysis by phagocytes and antifungal patients

Mycotoxicoses (diseases attributed to toxins from fungi) 1. Ingestion of mycotoxins ▪ Toxins coming from the Amanita mushroom (amanitin and phalloidin) - psychoactive and hepatotoxic 2. Ergot alkaloids ▪ Causes egotism or St. Anthony’s Fire ▪ Derived from secretions of Claviceps purpura (grows in grains such as rye, used in bread-making) ▪ Alkaloids - ergotamine and lysergic acid diethylamide (LSD) ▪ Effect - vascular and neurologic effects ▪ Development of gangrene and convulsions 3. Aflatoxins ▪ Toxins (coumarin derivatives) produced by Aspergillus flavus (yellow) ▪ Fungi grows if staple commodities are improperly stored (improper storage of grains, peanuts) ▪ Aflatoxins can enter the food chain infecting livestock (humans are infected when spoiled grains and peanuts are ingested) ▪ Can lead to hepatic carcinoma and liver damage ▪ Aflatoxin B1 - carcinogenic

Hypersensitivity Reactions • Allergies (manifested by asthmatic reactions mediated by IgE) ▪ Eosinophilia and wheal-and-flare skin test reaction • Due to immediate hypersensitivity response to spores Mycoses • Mycotic infections • Practical classification of fungi: 1. Superficial/Cutaneous Mycoses 2. Subcutaneous Mycoses 3. Systemic Mycoses 4. Opportunistic Mycoses • Pathogenesis ▪ Granuloma formation - formed during chronic inflammatory reactions; aggregates of activated macrophages ▪ Acute suppuration ▪ Tissue destruction

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Reduced cell-mediated immunity predisposes dissemination

LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS • Macroscopic • Microscopic • Biochemial reactions • Serological tests Specimen Collection and Handling • BSL 2 ▪ Always used for safety ▪ Also sufficient for mold cultures of Blastomyces dermatitidis, Cryptococcus neoformans, dermatophytes, Penicillium marnefferi, and Sporothrix schenckiii • BSL 3 - must be followed if working with Histoplasma capsulatum and Coccidioides immitis • Petri dishes are not recommended for fungal cultures; screw-top tubes are preferred Collection • Sterile (aseptic techniques should be observed) • Swabs are not recommended for filamentous fungi • Collect adequate and appropriate specimens • Submit and process immediately Transport • Transport at room temperature • Preferably within 2 hours • Overnight or 24-hour specimens should never be used, except for hair, nails, and skin • If a delay is expected (> 2 hours), the specimens should be stored at: ▪ 4° C ▪ 37° C • Antibiotics such as penicillin (20 U/mL), streptomycin (100,000 mg/mL), or chloramphenicol (0.2 mg/mL) can be added to prevent bacterial contamination Common Specimens • Hair - collect hair samples by clipping or plucking from the base of the hair shaft ▪ Large masses of hair are not necessary K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Wood’s lamp - may be used to identify some fungi from infected hair that glow upon illumination by the lamp Skin - samples are scraped from the outer edge of the surface lesion after disinfection with 70% alcohol ▪ KOH wet mount must be prepared to break tissue debris Nails - submitted as scrapings or cuttings in the whole nail ▪ Disinfect the skin with 70% alcohol ▪ KOH wet mount for deeper scrapings Abscesses and subcutaneous infections - obtain the exudates Respiratory specimens - sputum (deep cough collection); less viscous samples can be inoculated directly; BAL, bronchoscopy specimens Blood - may be collected in: ▪ Brain heart infusion ▪ Isolator tube (lysis centrifugation system) ▪ Septichek, Castaneda bottles (biphasic media) ▪ Automated Systems (BACTED, BacT/ALERT) ▪ Blood culture bottles (not recommended) Buffy coat - for diagnosis of histoplasmosis Bone marrow - heparinized; can be inoculated at the bedside CSF - 3rd tube is sent to the microbiology section ▪ Centrifuge to obtain a more concentrated specimen; filtration may also be done ▪ For India ink preparation and culture inoculation Eye - corneal scrapings; eye discharge Urogenital and fecal specimens ▪ May grow yeasts ▪ Centrifuge urine specimens first then inoculate onto media ▪ First-voided morning specimen preferred Tissues - should be minced, not ground; transported in sterile gauze with sterile NSS ▪

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Criteria for Specimen Rejection 1. Situation - no specimen identification on the container or a discrepancy on the request form and container label ▪ Action - do not accept the specimen; return to sender 2. Situation - sputum with > 25 squamous epithelial cells/LPF (i.e., criteria used in Bartlett’s classification) ▪ Action: recovery of pathogenic fungi is still possible provided that the specimen is inoculated onto a selective culture medium with antibiotics 3. Situation - dried-out swab or insufficient amount of specimen

Action: inform the sender to submit a new swab; generally, swabs are not recommended for fungal recovery, with the exception of yeasts from a mucosal surface 4. Situation - sample is submitted in an improper container or in an unsuitable condition ▪ Action: instruct the sender to submit a new sample. If not possible, process the specimen and indicate on the final report that the quality of specimen was compromised and that the result can be interpreted only in light of the clinical presentation 5. Situation - a 24-hour sputum/urine specimen for fungal culture is received ▪ Action: these specimens are suboptimal ▪

Direct Microscopic Examination • Provides a rapid report to the doctor • Can give clues about the pathogen’s identity • Provide evidence of infection even if it is negative in cultures • Examples: Gram stain, KOH, India ink, Histologic stains, Calcofluor white Gram Staining (Hucker modification) • Still the frontline staining method in the laboratory • Gram reactions ▪ Yeast, pseudohypha - Gram-positive (purple/blue) ▪ Hypha - pink Wet Preparation • Simplest method; uses sterile NSS (1-2 drops) • Used to observe yeast, hyphae, and pseudohyphae • Disadvantages: lacks contrast; difficulty in the differentiation and ID of fungal elements KOH Mount • Use of 10-20% KOH • For initial examination of keratinized tissue, tissues, thick mucoid specimens • Components ▪ KOH - clearing agent ▪ Glycerin - prevents drying • Steps ▪ Add a drop of KOH reagent to an aliquot of specimen ▪ Place coverslip and allow to stand for 20 minutes o Factors that hasten clearing - gentle heating, addition of 40% DMSO o Advantage - hair samples can be examined to determine if infection is endothrix or ectothrix K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Tease Mount • Use teasing needles to remove mycelia from culture • Placed on a slide and stained with Lactophenol Cotton Blue • Place coverslip KOH Calcofluor White • 10-30% KOH • 0.1% Calcofluor White ▪ Binds to chitin and cellulose ▪ Fluoresce under UV lamp (apple green or bluish-white) KOH with Lactophenol Cotton Blue • LPCB - enhances visibility of fungi ▪ Lactic acid - additional clearing agent ▪ Phenol - fungicide Blue-black ink/methylene blue • 2 parts KOH: 1 part of ink Calcofluor White - together with Evans blue Blankophor India ink (nigrosin) - for encapsulated fungi (Cryptococcus neoformans) Preparation of Mounts from Cultured Isolates • Scotch tape preparation ▪ Lactophenol cotton blue or aniline blue • Tease mount • Wet mount - coverslip is positioned over growth on agar before using pressure to disperse growth • Microslide culture - demonstrates natural morphology of fungal cultures Other Stains for Direct Examination • Giemsa/Wright’s stain - for diagnosis of histoplasmosis • Lactophenol cotton blue (Aman) - blue color; with 10% PVA • Periodic Acid Schiff (PAS) - presence of fungal hyphae (purplish red) • Gomori Methenamine Silver • Acridine orange • Masson Fontana • H&E • Mucicarmine (Southgate’s mucicarmine technique - for C. neoformans) • Toluidine blue O - Pneumocystis jirovecii Fungal Culture

Primary Culture Media a. Sabouraud’s Dextrose Agar - general purpose isolation medium; pH 6.5 inhibits bacterial growth ▪ Histoplasma and some Nocardia species will not grow ▪ Overlaid with olive oil for the growth of Malasezzia furfur b. Potato Dextrose or Potato Flake Agar - encourage growth of reproductive structures ▪ Stimulates growth of conidia ▪ Ideal for slide cultures c. Nutritionally poor media - used to stimulate production of reproductive structures ▪ Dilute Hay Infusion Agar, soil extract agar, 2% water agar d. Brain Heart Infusion Agar - primary recovery of saprobic and pathogenic fungi e. Brain Heart Infusion Biphasic Blood Culture Bottles - recovery of fungi from blood f. SABHI (Sabouraud Brain Heart Infusion) - primary recovery of saprobic and pathogenic fungi Selective Media a. Mycosel or Mycobiotic ▪ SDA with cycloheximide (inhibits fungal contaminants) and chloramphenicol (inhibits bacteria) ▪ For dermatophytes (skin, hair, and nail specimens) ▪ Inhibited pathogens: Cryptococcus, some Candida, and Aspergillus b. Inhibitory Mold Agar ▪ Contains chloramphenicol to inhibit bacteria ▪ Primary recovery of pathogenic fungi exclusive of dermatophytes c. Littman-Oxgall agar ▪ Selective general purpose medium to isolate fungi from contaminated specimens ▪ Inhibitors - crystal violet and streptomycin (inhibit bacteria), oxgall (prevents spread of fungal colonies) d. BHI with antibiotics - for pathogens exclusive of dermatophytes e. Yeast Extract Phosphate agar - primary recovery of pathogenic fungi exclusive of dermatophytes f. Smith’s medium (yeast extract phosphate agar with ammonia) ▪ Isolation and sporulation of Histoplasma capsulatum and Blastomyces dermatitidis from contaminated specimens

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Differential Media a. Dermatophyte Test Medium ▪ For Microsporum, Epidermophyton, and Trichophyton b. Ascospore agar - detection of ascospores in ascosporogeneous yeasts such as Saccharomyces c. Cornmeal agar with Tween 80 and trypan blue - for ID of Candida albicans by chlamydospore production and ID by microscopic examination d. Cottonseed conversion agar ▪ Converts mold phase of Blastomyces to yeast phase e. Czapek’s agar - for Aspergillus f. Niger seed agar (Birdseed agar)/Staib’s agar - for Cryptococcus neoformans ▪ Brown-black colonies in 4-7 days due to the breakdown of niger seed substrate by phenol oxidase to produce melanin ▪ Uses thistle (Guizotia seeds) g. Potato dextrose agar - demonstration of pigment production by T. rubrum and for preparation of microslide cultures h. Rice medium ▪ For identification of Microsporum audouinii (no growth on rice medium); Microsporum canis grows on rice medium i. Trichophyton agars (1-7) ▪ ID members of Trichophyton ▪ Nutritional requirement tests j. Urea agar ▪ Detects urease production by C. neoformans and T. mentagrophytes (differentiate with T. rubrum, which is ureasenegative) k. Yeast fermentation broth - ID yeasts by determining fermentation of various carbohydrates l. Yeast nitrogen base agar/Yeast assimilation medium - ID of yeasts by determining carbohydrate assimilation in the presence of oxygen m. Nitrate reduction agar ▪ Confirms nitrate reduction by Cryptococcus n. Cornmeal agar ▪ With 1% glucose ▪ D i f f e r e n t i a t e Tr i c h o p h y t o n r u b r u m a n d Tr i c h o p h y t o n mentagrophytes o. CHROMagar - selective and differential medium for clinically important yeasts ▪ Chloramphenicol inhibitor ▪ C. albicans - yellow-green to blue-green colonies

Incubation • Specimens should be incubated up to a month (4 weeks) and examined periodically before reporting as negative • Most fungi grow optimally at 30° C • Yeasts usually grow within 1-3 days while Histoplasma may require 10-12 weeks for growth • Petri dishes should be sealed with paraffin or scotch tape to enhance humidity Preservation of Cultures a. Storage in water - spores and conidia are washed with sterile water and placed in vials. Yeast culture can be transferred directly to sterile water in small vials sealed and stored at RT b. Freezing - at -70° C and placed in vials; pathogenic cultures should be placed in crushproof metal shipping containers before freezing c. Mineral oil - overlaid onto cultures; cap the tube tightly and store at RT d. Freeze drying Macroscopic Examination Pigment • Observe the reverse and obverse (surface) pigment of the culture • Take not if it is diffused or confined in an area • Dematiaceous - dark olive green to dark brown to black pigment • Hyaline - clear/colorless or pastel

Texture • Best observed in cross section; related to aerial hyphae and number of conidia/spores a. Glabrous - leathery/waxy; little if any aerial mycelium b. Velvety - resembles plush or velvet fabric or suede; have short aerial hyphae, few conidia or spores c. Yeast-like - resembles colonies of coagulase-negative staphylococci ▪ Bacteria-like ▪ Yeasts appear drier and duller ▪ No aerial mycelia d. Cottony - develop when colonies produce long aerial hyphae e. Granular - fungi that conidiate or sporulate heavily; powdery

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Topography - how the colony surface is arranged; best observed on the reverse side a. Flat - common; form is efficient and requires no extra effort or enzymes from the fungus b. Rugose - radial grooves or deep furrows that radiate from the center; like spokes of a bicycle wheel c. Folded - random folds; may be long, short, parallel, or at right angles d. Crateriform - least common; central depression surrounded by a raised edge e. Verrucose - wart-like or with rough knobs; wrinked; convoluted f. Cerebriform - brain-like g. Umbonate - button-like central elevation

Hair Penetration/Perforation Test - in-vitro test to distinguish • T. mentagrophytes (+) and T. rubrum (-) • M. canis and M. equinum • Positive result - V-shaped penetration

Growth Rate • Rapid growers - < 5 days • Intermediate growers - 6-10 days • Slow growers - 11 or more days or up to 8 weeks

Levodopa-Ferric Citrate Test • Phenol oxidase reacts with dihydroxyphenylalanine in the presence of ferric citrate to form melanin • Levodopa as substrate • Cryptococcus neoformans

Other Tests Germ Tube Test • Small amount of isolated yeast colony plus serum or plasma • Incubate at 37° C for 2-3 hours • A drop of suspension is examined microscopically • Positive - germ tube formation (Candida albicans); no constriction between germ tube and mother cell

Growth at 42° C • Differentiates Candida albicans (grows at 42° C) and Candida dubliniensis (does not grow at 42° C) which are both germ tube test-positive Serum Culture • Serum incubated with yeast cell at 37° C for 2-3 hours • If only yeasts are seen (negative - not Candida) • If there are yeast cells and hyphae - Candida • If yeasts, hyphae, and chlamydoconidia and germ tubes are present Candida albicans or Candida dubliniensis

Rapid Urease Test • For urease-producing yeasts recovered from respiratory specimens and other specimens • Positive - pink to purple color after 2 days (Cryptococcus neoformans) • Negative - no color change (Candida albicans) Rapid Nitrate Reduction Test - for Cryptococcus

Thiamine requirement - very useful for dermatophytes Growth on Rice Grains - differentiate Microsporum canis (+ for growth) and Microsporum audouinii (no growth after 10 days) Temperature Studies • Cryptococcus - growth at 35° C; no growth at 42° C • Candida albicans - growth at 42-45° C • Candida dubliniensis - no growth at 42° C

Serologic and Antigen Tests Complement fixation - for Histoplasma capsulatum and other dimorphic fungi Immunodiffusion test - for Histoplasma capsulatum ELISA - Aspergillus antibodies Cryptococcal Antigen in CSF and Serum Counterimmunoelectrophoresis Exoantigen tests

Hair Baiting Test - for dermatophytes since they are keratinophilic; dermatophytes will grow selectively on hair K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

SUPERFICIAL MYCOSES SUPERFICIAL MYCOSES •

• • •

Affects mainly the horny, non-living layer of the skin (stratum corneum) and extrafollicular parts of the hair ▪ Living tissues are not affected = host does not mount an immune response Innocuous (non-harmful) Non-dermatophytic May be tineas (skin infections) or piedras (hair infections) ▪ Tinea versicolor ▪ White piedra ▪ Black piedra ▪ Tinea nigra palmaris

TINEA VERSICOLOR • Synonyms: an-an, pityriasis versicolor, dermatomycosis fufuracea, tinea flava, chromophytosis, liver spot • S/S: a. Asymptomatic b. Hyperpigmented (in fair-skinned) or hypopigmented (in darkskinned) macules or patches c. Chronic: scaly d. May enlarge or coalesce e. Lesion: non-inflammatory, pruritic; covered with branny or furfuraceous scales, sharply marginated • Common sites: chest, upper back, shoulder, upper arms, abdomen ▪ Rarely in scalp, palms, feet Causative Agent • Caused by Malassezia furfur (Pityrosporium ovale/Pityrosporium orbiculare) • Yeast-like • Grows on the stratum corneum layer of the epidermis; may be part of NF • Lipophilic (affinity for lipids; add olive oil to SDA) • MOT: direct skin contact • High recurrence rate despite of treatment • Previously thought as a cause of dandruff

Laboratory Diagnosis A. Specimen a. Skin scrapings from discolored areas of the skin b. Blood or tissue samples if disseminated infection is suspected B. Macroscopic examination ▪ Wood’s lamp examination of lesions = golden yellow to light green fluorescence C. Direct microscopic examination of skin scrapings a. Alkali staining (CV, iodine, MB) b. KOH preparation: clusters of yeast-like cells with hyphal elements (spaghetti and meatballs appearance) D. Culture a. Sabouraud Dextrose Agar (SDA) overlaid with vegetable oil (peanut oil, olive oil), or whole fat milk o Incubation temperature: 35-37o C b. Macroscopic morphology of colonies: dry, smooth or lightly wrinkled; glistening or dull, white to creamy c. Microscopic morphology of colonies: hyphal structures are rare o Phialides (unicellular) = acquire collarettes during conidiogeny; resemble bowling pins or medicine capsules and bottles Risk Factors • Poor nutrition • Excessive sweating • Pregnancy • Cushing syndrome Treatment a. Whitfield’s ointment (salicylic acid + benzoyl peroxide) b. 2.5% selenium sulfide lotion c. 3% salicylic acid in 70% alcohol d. Sodium thiosulfate e. Azole family (ketoconazole, fluconazole, imidazole) f. Vinegar

PIEDRA • Means stone • Synonyms: Tinea nodosa, trichomycosis nodosa, trichomycosis nodularis, Beigel’s disease, Chignon disease • Chronic superficial infection of hair with nodular masses of fungal elements surrounding the hair shaft K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪ •

•

S/S

Base of hair shaft and hair follicle are unaffected

▪ Firm, irregular nodules or encrustations ▪ Multiple infections ▪ Hair shaft and follicle appear normal ▪ Base of the hair shaft and hair follicle unaffected MOT is unclear

BLACK PIEDRA • Piedraia hortae • Affected areas: scalp hair, eyebrows, eyelashes, pubic hair ▪ Infected hair is rough, sandy, or granular • Nodules ▪ Hard, fusiform, firmly attached to hair shaft ▪ Does not penetrate cortex of hair (ectothrix) ▪ Hair follicles not involved ▪ Colored black • Morphologic form: mold • Geographic site: tropics and subtropics Laboratory Diagnosis a. Direct Microscopic Exam (KOH) ▪ Nodules are composed of tightly packed, regularly arranged, thick-walled cells ▪ Hypha: dematiaceous ▪ Asci are found within the locules containing up to 8 ascospores ▪ Multiseptate hypha resembling arthrospores, many chlamydospores + ascospores b. Culture ▪ Compact, dark-brown to black, conical colonies with short aerial hyphae ▪ Grows slowly on SDA (2-4 weeks) at 25-30o C Treatment • Shave or cut hair • 2% selenium sulfide • Castellani paint • Amphotericin B lotion WHITE PIEDRA • Trichosporon beigelli complex • Affected areas: facial hairs, moustache, beard, hair of the axilla and groin • Morphologic form: yeast

•

Nodule ▪ ▪ ▪ ▪

White, cream to light brown Soft, mucilaginous, white, yellowish, beige Less adherent Discrete or more often coalescent, forming an irregular transparent sheath ▪ Hair follicles not affected • Geographic site: temperate zone Laboratory Diagnosis a. Direct Microscopic Exam (KOH) ▪ Often transparent, greenish, irregular sheath ▪ Cells are not as organized ▪ Blastospores and arthrospores adhering to the hair b. Culture ▪ Grows moderately on SDA (1-2 weeks) at 25-30o C ▪ Colonies: smooth, highly-wrinkled or radially folded, yeastlike, cream-colored; with mycelial fringe Treatment • Shave or cut hair • Terbinafine • Ketoconazole • Topical salicylic acid

TINEA NIGRA • Hortaea werneckii ▪ Formerly Exophiala werneckii, Phaeoannellomyces werneckii, Cladosporium werneckii ▪ Dematiaceous • Synonyms: keratomycosis nigricans palmaris, cladosporosis epidermica, pityriasis nigra, microsporosis nigra • Chronic, superficial, usually asymptomatic, fungal infection usually of the palms; common in farmers and other agricultural workers ▪ Other locations – sole of foot, interdigits, wrists, forearm, trunk, neck • Primary medical importance – often misdiagnosed as melanoma • S/S ▪ Usually asymptomatic ▪ Lesion – usually a dark patch on the palm of one hand with welldefined irregular margin about 1-5 cm in diameter ▪ Stained appearance (look like silver nitrate or India ink stains or functional type nevi) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Laboratory Diagnosis a. Specimen – skin scrapings from darkly pigmented cutaneous lesions b. Direct Microscopic Examination (KOH) ▪ KOH mount – long, sinuous, strongly dematiaceous, branching, septate hyphae and elongated budding cells ▪ Yeast-like cells are seen only after culture c. Culture ▪ SDA with antibiotics at 25-30o C ▪ Colonies: shiny, moist, yeast-like, dirty white to brown, covered with masses of conidia and budding cells o Will turn black in 2-3 weeks ▪ Microscopic – olive-black elliptical yeast-like cells (2-celled) o Annelloconidia are elliptical to subglobose (unicellular, hyaline to olive brown walls); larger than original annellides o Hyphal structures – thick-walled; septated with tapered terminal ends Epidemiology • Common in temperate and tropical countries • Agricultural workers (farmers, etc.) • More common in males Treatment • Whitfield’s ointment • Salicylic acid • Tincture of iodine • Azoles (ketoconazole, econazole, miconazole)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

CUTANEOUS MYCOSES CUTANEOUS MYCOSES • Dermatomycoses (dermatophytoses) ▪ Fungal diseases that infect skin, hair, and nails ▪ Keratinized skin layers and appendages ▪ Outgrowths of the epidermis (hair and nails) • Genera involved: a. Microsporum b. Trichophyton c. Epidermophyton • Involves a restricted area of the body • Tinea (ringworm) • Organisms are keratinophilic • Production of asexual spores ▪ Macroconidium – also known as fuseaux ▪ Microconidium – also known as microaleurispore • May be classified as: a. Geophilic – from the environment b. Zoophilic – from animals c. Anthropophilic – from humans Anthropophilic

Zoophilic

Microsporum canis

Microsporum gypseum

T. rubrum T. schoenleinii

Trichophyton verrucosum

T. violaceum

M. gypseum

M. canis

Macroconidia

Microconidia

Comments

Fusiform Thick-walled Distal end: thin filamentous tail Cigar-shaped

Tan/buff colonies

Thick-walled; spindleshaped; echinulate Spiny distal ends Beaked apex

Rapid grower Racquet and pectinate hyphae Bright yellow pigment on reverse side Emits fluorescence on infected area

Distorted cylinders; spindles with thick walls Chlamydoconidia

Clavate or pyriform (sparse)

Clavate or ovoid

Colonies: cottony white; velvety, rugose; light obverse pigment and reddish-brown reverse Pectinate vegetative hyphae Intermediate to slow grower Racquet hyphae Large vesicles

Trichophyton equinum Trichophyton mentagrophytes (granular isolates)

T. tonsurans

Species

M. audouinii

Microsporum audouinii

T. mentagrophytes

Microsporum • Infects skin and hair, rarely nails • Macroconidium – large, spindle-shaped, thick-walled, multiseptate (> 6 cells); echinulate; fusiform ▪ Rough, spiny (echinulate) and occurs at hyphal tip • Microconidium – few or absent • Colonies are tan or buff • Aerial mycelium – powdery of velvety

Geophilic

Epidermophyton floccosum

Trichophyton concentricum

MOT – close human contact; sharing of personal effects; contact with animals; fomites, soil-borne inoculum

Trichophyton • Infects skin, hair, and nails • Infection seen more commonly among adults • No fluorescence under Wood’s lamp • Macroconidia – smooth, club-shaped, thin-walled; with 8-10 septa • Microconidia – numerous; spherical; tear-shaped • Colonies – powdery, waxy, or velvety • All are slow growing, except T. Mentagrophytes (intermediate) and T. Tonsurans (slow to intermediate) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Species

Macroconidia

Microconidia

Comments

T. mentagrophytes

Thin-walled; smooth Cigar-shaped (4-5 cells)

Numerous Tear-shaped En grappe arrangement

Hypha: spiral; w/ nodular bodies Culture: flat and granular or downy colonies

T. schoenleinii

Rarely seen

Clavate, sessile

Favic chandelier (antler) vegetative hyphae Culture: glabrous, white to tan; heaped and folded Lesions with mousy odor

T. rubrum

Long, narrow, cylindrical (3-8 cells)

Clavate or pegshaped in clusters En thryses: in small clusters described as “birds on a fence” May form directly on macroconidia

Chlamydoconidia Hypha: pectinate Culture: white and downy w/ yellow to blood red reverse pigment

Clavate, pyriform, or ovoid Sessile

Culture: thin and white; glabrous; colorless or salmon Chains of macroconidia Sparse conidiation

Smooth parallel walls

T. verrucosum

T. tonsurans

Rat-tail

Short, blunt, irregular clubs Moderately thick smooth walls Rarely produced

Elongated with thin smooth walls

Truncate Balloon-shaped; clubshaped; tear-shaped Ballooning

Small, clavate

T. violaceum

May or may not be present

T. concentricum

Culture: flat, powdery with yellow brown to reddish brown pigment

May or may not be present

Production of chlamydoconidia Distorted twisted hyphae Swollen hyphal cells with cytoplasmic granules Sparse conidiation Culture: lavender colonies with verrucose surface and waxy texture Hyphae: broad, tangled, highly branged, septated with antler tips Culture: velvety, smooth, waxy, irregularly folded. White to yellowish or orange brown on reverse May produce short gray hypha

Epidermophyton • Infects skin and nails, rarely hair • Colonies: velvety or powdery with suede-like texture and yellow green or Khaki Green pigmentation • Macroconidia – smooth-walled • Microconidia – absent • With chlamydoconidia

TINEA •

•

Species

Macroconidia

Comments

E. floccosum

Snow shoe or snow paddle in pairs or triads Thin-walled

Slow-growing No microconidia

Ring worms – circular scaly patch of erythema with a raised border ▪ Edges are more inflamed ▪ Usually localized but may disseminate among immunocompromised patients Risk factors: ▪ Age (Tinea capitis among children; onychomycosis among elderly) ▪ Family history ▪ Participation in sports activities with extensive body contact ▪ Barefoot use of communal aquatic facilities (Athlete’s foot) ▪ Sharing of personal effects (towel, hairbrush) ▪ Living in rodent-infested houses ▪ Contact with infected animals

Tinea Capitis – ringworm of the scalp • Etiologic agents ▪ M. audouinii – formerly the most common cause ▪ M. canis ▪ T. tonsurans – now the most common cause • Clinical forms: a. Gray patch ringworm o Common among small children o Seldom inflamed o Ectothrix pattern of hair invasion K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

o M. audouinii and M. canis o Usually contracted by sharing combs b. Black dot ringworm o Endothrix pattern of hair invasion o Black dot stubs because of brittle infected hair o T. tonsurans and T. violaceum c. Inflammatory ectothrix infection - caused by T. mentagrophytes d. Kerions - produced by T. tonsurans Tinea Favosa – favus honeycomb ringworm, scalp hair ringworm • More severe form of tinea capitis seen among Scandinavians • Synonyms: favus • Etiologic agent – T. schoenleinii • Manifestations: ▪ Scrutula (yellow cup-shaped crust) around infected hair follicle; yellow encrustations; may lead to permanent alopecia ▪ Infect scalp exudes a mousy odor Tinea Barbae – ringworm of the beard; affecting the bearded areas of the face and neck; Barber’s itch • Etiologic agents ▪ T. rubrum ▪ T. violaceum ▪ T. mentagrophytes ▪ T. verrucosum • Men in close contact with animals are at risk • Synonyms – Barber’s itch, trichophytosis barbae, tinea sycosis, pseudofolliculitis barbae

•

Clinical forms: a. Superficial, non-inflammatory – resembles bacterial folliculitis ▪ T. rubrum, T. violaceum b. Deep, inflammatory – deep, kerion-like plaques ▪ T. mentagrophytes, T. verrucosum

Tinea Corporis – ringworm of the body • Affects the non-hairy portions of the body (glabrous skin) • Reddened, circular scaly patch with sharply demarcated margin • Etiologic agents: T rubrum, T mentagrophytes

Tinea Cruris – ringworm of the groin; Jock’s itch; eczema marginatum, Dhobie itch, tinea inguinalis, Groin dermatophytosis, junk rubbin, gym itch, Crotch rot, camper rash, The chaffin • Pruritic fungal infection affecting both sides of the inner thigh and proceeds downwards away from the genitocrural folds • Etiologic agents – T rubrum, T mentagrophytes, E floccosum Tinea Pedis – ringworm of the foot; Athlete’s foot (alipunga) • Affects the interdigitating areas and sole of the foot • Scaly patches of itchy, inflamed skin • Most common type of fungal infection • Etiologic agents – T mentagrophytes, T rubrum, E floccosum • Clinical forms: a. Intertriginous – most characteristic type; between toes/skin folds b. Moccasin – entire sole, heel, and sides of the foot c. Bullae – sides of the foot/instep Tinea Unguium – nail ringworm; onychomycosis; dermatophytosis of the nails • Thickening, brittleness, and dystrophy of the nails • Usually begins at the edges, then spreads • Yellow, brittle, and thickened nails • Etiologic agents – T rubrum, T mentagrophytes, E floccosum Tinea Manuum – ringworm infection of the skin of the hand • Palms are dry, scaly, hyperkeratotic • Etiologic agents – T rubrum, T mentagrophytes, E floccosum Tinea Imbricata – scaly ringworm of the glabrous skin; Tokelau ringworm, Trichiophyta corporis superficialis, Malabar itch, Oriental ringworm, Beauty ringworm, Herpes farinosus or herpes desquaman, Tamana, Tinea circinata tropicalis, Gugo • Skin eruptions consisting of a number of concentric rings of overlapping scales forming papulosquamous patches scattered to any part of the body • Common in the Pacific Polynesian Islands • Etiologic agent – T concentricum PATHOGENESIS • Dermatophytes can degrade keratin (keratinases) • Keratophilic fungi – use keratin as a substrate • Release of keratinases = cause disturbance of epidermal barrier LABORATORY DIAGNOSIS • Specimen – skin scrapings K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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KOH mount – 10%, 20% Hair samples – broken, twisted, or scaly (use petri dish or paper) Wood’s Lamp – no fluorescence in M. gypseum and Trichophyton ▪ The rest exhibit yellow fluorescence Culture – SDA with cycloheximide, chloramphenicol DTM (Dermatophyte Test Medium) o DTM indicator – yellow to become red at RT (pH indicator = phenol red) o Incubate for 14 days o (+) – presence of dermatophyte (alkaline) Rice Grain Medium o M. canis – (+) o M. audouinii – (-) o 10-14 days Urease test o T. mentagrophytes – (+) in 2-3 days o T. rubrum – (-) Hair baiting test o T. mentagrophytes – (+) after 7-10 days o T. rubrum (-) o 10% yeast extract Cornmeal agar (with 1% glucose) o T. rubrum – red pigment o T. mentagrophytes – no pigment Hair penetration test o T mentagrophytes (+); T rubrum (-)

TREATMENT A. Azoles ▪ Miconazole, clotrimazole, econazole ▪ Topical ▪ MOA: interferes with cytochrome P450-dependent enzyme systems at the demethylation step from lanosterol to ergosterol (prevent sterol synthesis) B. Griseofulvin ▪ Fungistatic; affects the microtubular system offungi ▪ Interferes with the mitotic spindle and cytoplasmic microtubules ▪ Oral

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

SUBCUTANEOUS MYCOSES SUBCUTANEOUS MYCOSES • Also known as inoculation mycoses • Fungal diseases that result from traumatic skin puncture from thorns or vegetation • Usually confined to subcutaneous tissues (or lymph nodes) • Diseases are usually chronic in nature • At risk ▪ Males in Mexico ▪ Farmers • Granuloma formation is common • Lesions are usually found on the extremities • All agents are dematiaceous, except Conidiobolus coronatus (hyaline; aseptate) • Includes the following diseases: 1. Spototrichosis 2. Lobomycosis 3. Rhinoentomophthoromycosis 4. Mycetoma 5. Chromomycosis 6. Phaeohyphomycosis 7. Rhinosporidiosis

SPOROTRICHOSIS • Also known as: Rose Gardener’s disease, Rose Handler’s disease • Chronic infection of the cutaneous tissue, subcutaneous tissue, and lymphatics • Causative agent: Sporothrix schenckii complex • MOT: usually occurs from skin trauma due to accidental pricking; inhalation of the fungus may also lead to pulmonary infections • Affected body parts - upper and lower limbs Clinical Manifestations • Mostly seen as lymphocutaneous sporotrichosis ▪ Papules at the portal of entry can ulcerate and may spread via the lymphatic system

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Other forms: ▪ Fixed cutaneous sporotrichosis - painless; characterized by exudation ▪ Pulmonary sporotrichosis ▪ Extracutaneous forms Pathology - granuloma formation in tissues

Diagnostic Features • Splendore-Hoeppli phenomenon (asteroid bodies) seen in tissues ▪ Radiating eosinophilic structures (pinkish) ▪ Manifestation of the body’s immune response to the organism • The fungus is dimorphic ▪ Yeast - cigar-shaped ▪ Mold - flowerette or daisy-head appearance Laboratory Diagnosis • Specimen - skin biopsy, aspirate, pus, or curetting • KOH mount/stains (look for cigar-shaped bodies) Treatment • Potassium iodine (oral) • Itraconazole

LOBOMYCOSIS • Chronic, localized sub-epidermal infection causing keloids blastomycosis • Causative agent: Lacazia loboi (formerly Loboa loboi) • Humans and dolphins can be affected • Endemic area: Amazon river basin (Brazil) • More common in males Clinical Manifestations • Initial infection - traumatic inoculation in cases of insect stings/bites, snake bite, stingray sting, or in cutting vegetation • Disease manifestations ▪ Chronic, localized, subepidermal infection ▪ Presence of keloids, verrucoid, nodular lesions ▪ There may also be the presence of vegetating crusty plaques and tumors • Lesions appear in the face, arms, legs, or ears • Autoinoculation

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Laboratory Diagnosis • Specimen: skin scrapings, biopsies, exudates • Direct examination ▪ KOH mount with Parker Ink of Calcofluor white ▪ Stains: PAS, GMS, Gram stain ▪ Staining with GMS will show numerous darkly pigmented yeast cells, often in chains (dematiaceous) • Culture - currently not possible Treatment • Surgical excision - complete removal • Clofazimine (partially effective)

RHINOENTOMOPHTHOROMYCOSIS • Chronic inflammatory or granulomatous disease restricted in the nasal mucosa • Causative agent: Conidiobolus coronatus/ Entomophthora coronata (a Zygomycete) • Cases are seen in the tropical rain forest areas of central, west and south America Clinical Manifestations • Characterized by polyps or palpable restricted subcutaneous tissues (in the nose) • Symptoms - nasal obstruction, drainage, sinus pain • Progressive generalized facial swelling may occur (disfiguring tissue mass) Laboratory Diagnosis • Specimen: skin biopsy, needle biopsies from pulmonary lesions, skin scrapings • Direct examination ▪ Skin scrapings: KOH with Parker Ink of Calcofluor white ▪ Tissue: stains such as GMS and H&E ▪ Result: numerous branching, aseptate hyphae • Serology ▪ Highly sensitive and specific (immunodiffusion) • Culture - SDA without cycloheximide ▪ Cycloheximide kills saprophytes (Zygomycetes are saprophytic) ▪ Fast growing, grey or white, downy colonies

Treatment • Surgery • KI • Amphotericin B

RHINOSPORIDIOSIS • Chronic, usually painless infection, characterized by the development of mucosal polyps in the nasal mucosa and conjunctiva • Causative agent: Rhinosporidium seeberi (now considered a protozoan) • Habitat of the organism is unknown but swimming in lakes, ponds, and rivers is linked to the disease Clinical Manifestations • Affected areas: nose, nasopharynx, soft palate, and occasionally, the genitals • Most cases are reported in India and Sri-Lanka • Respiration may be compromised due to nasal polyps • Sporangia may be grossly visible (diagnostic feature) Laboratory Diagnosis • Biopsy ▪ Histopathology/KOH ▪ Presence of thick-walled cysts (sporangia) ▪ May appear as white dots in mucosa Treatment • Surgery • Dapsone (for relapsed cases)

MYCETOMA • Also known as Madura foot, Maduromycosis, or Watering Can Foot • Chronic skin and subcutaneous skin infection • Occurs after a traumatic injury with a contaminated object such as a thorn or splinter • Caused by a variety of organisms such as fungi (eumycotic mycetoma) or bacteria (actinomycotic mycetoma) • Eumycotic mycetomas ▪ Madurella mycetomatis (most common) ▪ Madurella grisea, Exophiala jeanselemei, Acremonium falciforme, Pseudollescheria boydii K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• •

Actinomycotic mycetomas ▪ Actinomadura, Nocardia, Streptomyces Botyromycotic mycetoma (pseudomycetoma) ▪ S. aureus, Bacteroides, E. coli, P. aeruginosa

Clinical Manifestations • Characterized by swelling and suppuration of subcutaneous tissue and formation of sinus tracts with extension to the bone • Granules (grains) are seen (aggregates of the organism) in the draining pus ▪ Black granules (pigmented) and yellow granules (non-pigmented) • Affected areas: hands or feet Epidemiology • Most commonly found in Central and South American, Africa, and India • Outdoor laborers are the most effected population • No person-to-person transmission Laboratory Diagnosis • Demonstration of granules in a tissue biopsy, draining exudates, aspirates • Direct observation (Gridley stain) • Grains - broad, interwoven septet hyphae ▪ Black grains - dematiaceous ▪ White grains - non-pigmented • Culture - SDA Madurella • Most common cause of eumycotic mycetoma • Dematiaceous fungus • Produce black granules • M. mycetomatis - produce conidia from the tips of phialides; many remain sterile ▪ possesses a diffusible brown pigmented • M. grisea - sterile hypha are seen; produce a reddish-brown pigment Treatment • Surgery • Azoles (itraconazole, or voriconazole)

CHROMOMYCOSIS • Also known as chromoblastomycosis, Mossy Foot disease, dermatitis verrucosa, hematomycosis

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Causative agents: Fonsecaea pedrosoi (most common), Cladophialophora carrionii, Phialophora verrucosa, Fonsecaea compacta, and Rhinocladiella aquaspersa Dematiaceous

Clinical Manifestations • Chronic mycosis of the cutaneous and subcutaneous tissue that develops over time (months and even years) • Development of dematiaceous planate dividing rounded sclerotic bodies in infected tissues ▪ Diagnostic feature - brown and round cells (sclerotic bodies) • Lesions appear as crusted, ulcerated, verrucose, and wart-like ▪ Long-standing lesions appear cauliflower-like • May also appear as mycetomas, but there is no bone involvement • Lesions appear in the extremities (feet and lower legs) Epidemiology • Distribution: worldwide but most cases are seen in tropical and subtropical areas • Most common: Fonsecaea pedrosoi Laboratory Diagnosis • Specimen: skin scrapings/biopsy • Direct examination ▪ KOH with Parker Ink or Calcofluor White (scrapings) ▪ Stains (PAS, GMS, and H&E) for tissue specimens ▪ Presence of sclerotic bodies (arrangement of conidia and the manner on how the conidia are borne) • Culture: SDA Treatment • Surgery • Flucytosine with or without thiabendazole • Itraconazole

PHAEOHYPHOMYCOSIS • Fungal disease caused by dematiaceous septet fungi • Causative agents: Exophiala jeanselmei, Wangiella dermatitidis, Cladosporium, Cladophialophora bantiana, Phialophora, etc.

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Clinical Manifestations • Occurs worldwide; infection occurs after inoculation of contaminated wood splinter, thorns, or from soil • Formation of solitary asymptomatic nodule or cyst in immunocompromised patients • Disseminated disease seen in immunocompromised patients (brain abscesses) Laboratory Diagnosis • Specimen: skin scrapings, tissue biopsy, bronchial washings, CSF, pleural fluid • KOH mount - for skin scrapings, bronchial washings • Stains (GMS, PAS, and H&E, Masson-Fontana ▪ Presumptive for diagnosis - irregular swollen septet hyphen elements with yeast-like structures • Culture - SDA Treatment • Same as chromomycosis

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

1.

SYSTEMIC MYCOSES • GENERAL CONSIDERATIONS for DIMORPHIC SYSTEMIC MYCOSES • Systemic mycoses are caused by dimorphic fungi ▪ Yeast form (spherule in certain species) – at body temperatures ▪ Mold form – at room temperature (plate/environment) • Possess the ability to bypass the body’s defenses by changing their morphological form (dimorphism) • Also called primary mycoses (infection in both healthy and immunocompromised)/deep-seated mycoses • Pathogens primarily inhabit the soil ▪ MOT – inhalation of spores • Occurrence is rare; diseases are limited and endemic to certain geographic areas ▪ Ongoing transmission of infection in a particular area • Primary site of infection – lungs (pulmonary), then disseminate to other areas via blood or lymph • Probability of dissemination is high • Most dimorphic fungi are laboratory hazards ▪ Cultures of B. dermatitidis, C. immitis, and H. capsulatum – severe biohazard to laboratory personnel and must be handled with extreme caution in an appropriate pathogen handling cabinet ▪ Major biohazard to laboratory personnel – Coccidioides immitis • Affect immunocompetent individuals • Each fungal agent has a well-defined endemic area • Culture: a. Mold phase: SDA, Potato Dextrose Agar, SDA w/ antibiotics b. Yeast phase: BHI, BHI w/ blood, Pine’s, Kelly’s • Host response – cell-mediated immune response (granulomatous) • Treatment – amphotericin B and azoles

BLASTOMYCOSIS: Blastomyces dermatitidis • Synonyms: Gilchrist’s disease, North American blastomycosis, Chicago disease • Chronic infection found mainly in the lungs with suppurative and granulomatous lesions with characteristic skin lesions; may also spread to bone, but 70% of cases have skin dissemination • Predilection:

Men – due to occupation/recreational activities; may also be due to hormones (testosterone promote conversion from mold to yeast; estrogen in females prevents conversion from mold to yeast) 2. Persons who have more than normal contact with soil IP: 4-6 weeks

Clinical Manifestations A. Pulmonary ▪ Acute and pulmonary ▪ Radiographically, it may mimic PTB ▪ S/S: flu-like B. Extra-pulmonary (due to lymphatic dissemination) ▪ Granulomatous lesions and abscesses often occur ▪ Skin and bones, genitourinary blastomycosis (prostate), hematogenous spread to brain ▪ Lesions are raised, wart-like/verrucose; near the nose Epidemiology • Primarily endemic in Mississippi River Valley basin ▪ Less commonly in Africa, parts of Central and South America • Reported also in other parts of the world • Reservoir: soil near rivers ▪ Factors: acidic pH, high nitrogen and organic temperature; high soil temperature = promote growth of organism Laboratory Diagnosis • Specimens – sputum, BAL, bronchial biopsies, skin biopsies, prostatic secretions • Laboratory tests – direct microscopy, EIA, molecular methods (WI-1 gene), culture, antigen detection (A antigen, WI-1 antigen) • Tissue form (from tissue biopsy) – grows as thick-walled yeast cells with broad-based bud • Mold phase – microconidia (oval or pyriform) ▪ Lollipop (or dumbbell) microconidia • Culture: SDA; growth within 2-3 days ▪ At 25-30o C – fluffy white colonies to glabrous tan colonies ▪ At 37o C – colonies: yeast cells; hyaline; smooth-walled; thickwalled o For yeast culture, add cysteine to promote conversion of mold to yeast ▪ Cottonseed conversion agar – conversion of dimorphic fungus B. dermatitidis from mold to yeast form K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

PARACOCCIDIODOMYCOSIS: Paracoccidioides brasiliensis • Synonyms: South American blastomycosis, Brazilian blastomycosis, LutzSplendore-Almeida disease, paracoccidioidal granuloma • MOT: inhalation of conidia • Chronic granulomatous disease (skin, lymph nodes, mucous membranes, internal organs) ▪ Lungs – initial site of infection (usually asymptomatic) ▪ Lymph node involvement ▪ Ulcerative mucocutaneous lesions on face, mouth, nose ▪ Lesions are seen on the cooler areas of the body (nasal mucosa as the site of dissemination via lymph) • Disease has a long latency ▪ 10-20 years before disease manifestation ▪ Unknown incubation period • Risk factors – contact with soil + travelling to Brazil ▪ Males are also more at risk • Travel history to endemic areas is important in diagnosis • Epidemiology ▪ Endemic in South America, especially Brazil ▪ Organism resides in the soil Laboratory Diagnosis • Specimens: lower respiratory samples, specimens coming from lesions, lymph nodes, adrenal glands • Laboratory tests – direct microscopy, molecular methods (gp43 gene), culture, serology (CF, immunodiffusion), exoantigen test (serologic test for dimorphic fungi) • Tissue phase (from tissue biopsy) – translucent walled yeast cells with multiple buds ▪ Mariner’s wheel appearance or Mickey Mouse cap appearance • Mold phase – small one celled conidia (microconidia) similar to B. dermatitidis; borne on septate hyphae • Culture: ▪ At 25-30o C – colonies are flat, glabrous to leathery, wrinkled to folded, floccose to velvety, pink to beige to brown with a yellowish-brown reverse ▪ At 37o C – yeast cells Treatment • Amphotericin B • Itraconazole

COCCIDIOIDOMYCOSIS: Coccidioides immitis • Synonyms: San Joaquin Valley Fever, desert fever, desert rheumatism, cocci, the bumps • Most virulent of all fungal infections • Etiology: Coccidioides immitis or Coccidioides posadasii ▪ Both organisms are very similar morphologically ▪ Differ in endemic area o C. immitis – San Joaquin Valley area o C. posadasii – outside San Joaquin Valley • MOT: inhalation of arthroconidia Clinical Manifestations • Respiratory/pulmonary infection that may progress to a chronic or systemic disseminated disease involving the meninges, bones, joints, subcutaneous and cutaneous tissues • Hypersensitivity reactions – delayed hypesensitivity ▪ Occurrence of Valley Fever (40% of newly infected) o Valley fever – flu-like illness after inhalation of spores ▪ Presence of erythema nodosum and erythema multiforme = increased chances of dissemination o Erythema nodosum – seen among females; the bumps ⬥ Reddish, warm, tender (painful when palpated) in legs ⬥ Due to inflammation of subcutaneous tissues ⬥ Due to a delayed hypersensitivity reaction o Erythema multiforme – seen among children ⬥ Seen in arms of children ⬥ Multiple reddish, round lesions on the affected parts of the body ⬥ Target lesions ⬥ Due to the deposition of IgM immune complexes to the blood vessels on the skin = trigger complement activation = inflammation of microvasculature ▪ Toxic erythema ▪ Arthritis •

Disseminated forms: ▪ Common areas: skin, skeletal system, meninges ▪ Skin: papules, ulcers, abscesses ▪ Skeletal system: arthritis (desert rheumatism) ▪ Meningitis – most serious form K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Epidemiology • Endemic in Southwestern US (desert areas) – San Joaquin Valley, Southern Arizona, Utah, Nevada, New Mexico, Western Texas, Central and South America • Ecology: dry desert alkaline soil • Highly virulent: low infective dose • Outbreaks: seen after windstorms and earthquakes • More common during the summer and fall seasons • Immunocompromised persons – more prone to disseminated forms ▪ Race: Filipinos and African-Americans have high incidences for the disseminated form ▪ Sex: more common in men ▪ Pregnancy: pregnant women have the same risk as men o Progesterone and estradiol promote transition from mold to yeast • Already classified as bioterror agents Laboratory Diagnosis • Specimens: skin scrapings, sputum and bronchial washings, CSF, pleural fluid and blood, bone marrow, urine, and tissue biopsies from various visceral organs • Laboratory tests: direct microscopy, EIA, molecular methods (Antigen 2 [Ag2] or PRA [Proline-rich antigen] gene), culture, serology • Tissue form (37o C) – presence of thick-walled spherules ▪ Spherules – thick-walled structure containing endospores ▪ PAS – best stain to demonstrate spherules • Mold form – barrel-shaped arthroconidia ▪ Dysjunctor cells between arthroconidia • Culture: growth in 1 week ▪ 22o C – white-gray, moist, glabrous Treatment • Amphotericin B (for disseminated cases) • Azoles (less severe forms)

HISTOPLASMOSIS: Histoplasma capsulatum • Synonyms: Darling’s disease, reticuloendothelial cytomycosis, Cave disease, spelunker disease, African histoplasmosis • Etiology: a. Histoplasma capsulatum var. capsulatum

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b. Histoplasma capsulatum var. duboisii MOT: inhalation of microconidia

Clinical Manifestations • Intracellular mycotic infection of the reticuloendothelial system ▪ Inside macrophages • Most cases are asymptomatic and limited (95%) ▪ Smokers with COPD (chronic obstructive pulmonary disease) – severe infection • Acute pulmonary infection • May lead to chronic pulmonary infection ▪ TB-like symptoms; pulmonary neoplasms ▪ Granuloma formation ▪ Cavitations, fibrosis • Dissemination ▪ RES ▪ Hepatic infection ▪ Adrenal gland destruction ▪ Skin and mucosal surfaces (mouth and throat) ▪ Brain ▪ GI tract • Mucosal ulcerating lesions • Other areas affected: CNS, GI tract Epidemiology • Occurs worldwide • Major endemic area: Mississippi River Valley basin (US) ▪ Major river valleys in North and South America • For African histoplasmosis – central Africa • Also found far east – at least 50 countries in the tropics and temperate zones including the Philippines • Ecology: organism resides exclusively in soil containing guano of certain birds (starlings, chicken, black birds) and bats ▪ Soil rich in nitrogen ▪ Bat caves have high deposits of guano; cave exploration may result in exposure to high levels of the organism ▪ Birds and bats are not affected Laboratory Diagnosis • Specimens – sputum, BAL, blood (using Isolator), urine, lymph node, BM samples, CSF (meningitis) ▪ Isolator system – lysis + centrifugation; isolate cells inside macrophages K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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•

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Laboratory tests – direct examination, antigen detection (HPA: Histoplasma polysaccharide antigen), molecular methods, culture, serology (histoplasmin skin test, exoantigen test, CF) Tissue form – small, non-encapsulated intracellular yeast cells ▪ May be mistaken as amastigotes or Donovan-Leishman forms of Leishmania spp. Mold form – tuberculate macroconidia (with protruding structures) Culture: fluffy white colonies after 10-14 days ▪ SDA, BHI, BAP, Smith and Goodman’s medium, Yeast extract with ammonium hydroxide o Smith and Goodman’s medium (Yeast extract phosphate agar with ammonia) – for highly contaminated samples; more antibiotics ▪ BHI at 37o C – convert from mold to yeast

Treatment • Amphotericin B in severe infections • Itraconazole AFRICAN HISTOPLASMOSIS: H. capsulatum var. duboisii • Mycotic infection that localizes in skin, lymph nodes and bone, producing subcutaneous abscesses and skin lesions ▪ Multiple, pale, nodular granulomatous lesions of the skin in disseminated infections • Tissue form – large yeast cells inside giant cells; double cell or figure eight appearance • Culture ▪ RT: SDA (mycelial form with tuberculate macroconidia and microconidia) ▪ 35-37o C: BHI (yeast cells; larger than the yeast cells of H. capsulatum var capsulatum)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

OPPORTUNISTIC MYCOSES OPPORTUNISTIC MYCOSES • •

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•

Fungal infections usually seen in immunocompromised (AIDS, chemotherapy) or debilitated patients (hospitalized, with chronic diseases) Etiologic agents are saprophytic fungi which are not usually virulent ▪ Not typically a concern for human disease ▪ Immunocompromised individuals are affected ▪ Increasingly common due to various treatment regimens and immunosuppressive diseases (AIDS) Predisposing factors: a. Neutropenia b. Defective cell-mediated immunity c. Use of broad spectrum antibiotics d. Intravascular catheters (nosocomial) e. Chemotherapeutic agents f. Debilitating illness Common opportunistic fungi: ▪ Cryptococcus neoformans ▪ Candida albicans ▪ Pneumocystis jiroveci ▪ Aspergillus spp. ▪ Mucor/Rhizopus spp.

Candida • Disease: candidiasis, moniliasis, thrush, mycotic vulvovaginitis, Candida perionychia, Candida endocarditis ▪ Most notorious agents of yeast infection ▪ Most common cause of fungemia • Primary or secondary mycotic infection caused by members of the genus Candida • NF: mucosa, skin, digestive tract ▪ Cause disease in any site when host conditions are altered • Species: a. C. albicans – 50% of cases; premier cause of yeast infection in the world b. C. glabrata – second most common; 21% of cases

c. d. e. f. g.

C. parapsilosis – major cause of nosocomial infections C krusei C tropicalis C lusitianiae C dubliniensis

Candida albicans • Major cause of candidiasis • NF: mouth, rectum, vagina, skin ▪ May also be isolated from environmental sources (soil, water, plants) • Clinical manifestations: a. Cutaneous b. Vulvovaginal c. Oropharyngeal d. Conjunctivitis e. Gastrointestinal f. UTIs g. Invasive candidiasis (candidemia, meningitis, pneumonia) Clinical Manifestations A. Cutaneous candidiasis ▪ Intertriginous candidiasis – commonly seen in axilla, groin, submammary folds, umbilicus, interdigital spaces o Moist, erythematous macular rash o Friction promotes infection ▪ Diaper candidiasis – nappy rash; infants o If diapers are not changed ▪ Paronychia (painful) and onychomycosis (complete destruction of nails) – nails o Bakery and laundry workers are at risk B. Vulvovaginal candidiasis ▪ Also known as vaginal thrush o At risk: sexually active, diabetics, pregnant (GDM), those taking antibiotics ▪ S/S: pruritus, burning sensation, vaginal discharge (may be creamy and curd-like), dysuria ▪ Balanitis (in males) – inflammation of the glans penis C. Oropharyngeal candidiasis ▪ Thrush (oral thrush) o Immunocompromised (AIDS, diabetes) ▪ Appearance: white plaques (milk-curd appearance) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

D. Conjunctivitis ▪ Seen in people fond of using eyedrops containing steroids (immunosuppressant) E. Gastrointestinal candidiasis ▪ Esophagitis F. UTIs G. Invasive disease ▪ Candidemia – in blood ▪ Meningitis ▪ Pneumonia Laboratory Diagnosis • Specimens: ▪ Skin and nail scrapings ▪ Urine, sputum, and bronchial washings ▪ CSF, pleural fluid, and blood – for invasive infections ▪ Tissue biopsies from various visceral organs and indwelling catheter tips • Direct examination ▪ KOH ▪ GMS if in tissue ▪ Gram stain ▪ Calcofluor white ▪ PAS • Diagnostic features in direct examination: 1. Budding yeast cells 2. Pseudohyphae 3. Chlamydoconidia (thick and round spores) especially on Cornmeal agar • Culture: white to cream with a smooth, glabrous to waxy surface • Other tests: a. Germ Tube test: (+)-C. albicans and C. dubliniensis b. Growth at 42o C: (+)-C. albicans; (-)-C. dubliniensis c. Yeast morphology on Cornmeal agar d. Yeast assimilation tests • Serology: ELISA, CIE, RIA Treatment • Azoles, nystatin, amphotericin B • Resistance reported especially in non-Candida albicans species

Cryptococcus • Disease: cryptococcosis, torulosis, European blastomycosis, Busse-Buschke disease • Agents: a. Cryptococcus neoformans o Opportunistic – serotypes A and D o Primary pathogens – serotypes B and C o Usually seen in immunocompromised patients (AIDS patients, lymphoma, ALL, CLL, corticosteroid therapy) o Reservoir – pigeon guano (droppings) ⬥ Found worldwide o Encapsulated (polysaccharide; virulence factor) b. Cryptococcus gattii o Usually seen in immunocompetent patients o Reservoir – environment; Eucalyptus trees (Red Gum tree) o Endemic areas – tropics and subtropics (Northern Australia, Papua New Guinea, Zaire, Brazil, Venezuela, Southern California) • MOT: inhalation of basidiospores (sexual spores) and/or desiccated yeast cells • Causes systemic infection in both immunocompetent and immunocompromised patients Clinical Manifestations • Subacute or chronic infection most frequently involving the tissues of the CNS, but occasionally producing lesions in the skin, bones, lungs, or other internal organs • Predilection: lungs and CNS A.

Respiratory – pulmonary disease ▪ Can disseminate to other parts of the body B. Cutaneous – single or multiple papules that enlarge and ulcerate C. Bone and joint infections D. Cryptococcal meningitis ▪ Most frequent and most serious ▪ Headache, changes in mental status and personality, low grade fever ▪ Typical signs of meningitis (stiff neck, Kernig’s and Brudzinski’s signs) are absent K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

o o

Kernig’s sign – knee pain Bruzsinski’s sign – nuchal pain

Laboratory Diagnosis • Specimen - CSF • Direct examination ▪ CSF and pulmonary tissue mounted in 10% KOH or India Ink ▪ Stains: PAS, GMS, H&E, Mucicarmine ▪ India ink preparation – demonstrate capsule; halo appearance ▪ Southgate’s Mucicarmine Technique – demonstrated capsule appears red • Culture ▪ SDA (Mycosel may inhibit Cryptococcus) ▪ Birdseed agar (Niger’s seed agar) – brown-black colonies after 4-7 days due to the breakdown of niger seed substrate by phenol oxidase to produce melanin ▪ BHI • Serology (test for cryptococcal antigens) • PCR • Urease ▪ Use of Urea agar ▪ Indicator – phenol red ▪ (+) – fuchsia color of medium (C. neoformans) Treatment • For CNS involvement, best treatment is amphotericin B with fluorocytosine for 6-10 weeks or > 2 weeks amphotericin B + 5 fluorocytosine then fluconazole for at least 10 weeks

Aspergillus • Disease: aspergillosis ▪ Mycotoxicosis (aflatoxin) – ingestion of peanuts ▪ Allergy and sequelae due to the presence of conidia ▪ Colonization of preformed cavities or debilitated tissues ▪ Invasive, inflammatory granulomatous narcotizing lung disease ▪ Systemic and disseminated disease • Etiologic agents a. A. fumigatus – ubiquitous; infections of skin, ears, and other organs b. A. flavus – seen in patients with fungal sinusitis and keratitis c. A. ferreus – propensity for dissemination

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d. A. niger Disease usually seen in immunocompromised patients Neutropenia – single most predictive factor for disease development MOT – inhalation of conidia, inoculation, ingestion

Clinical Manifestations • Rarely cause disease among healthy persons, but cause serious disease in the immunocompromised A.

Respiratory – initial and most usual site of infection ▪ Respiratory tract (paranasal sinuses and lungs) ▪ Fungal sinusitis (A. fumigatus) ▪ MOT: inhalation B. Keratomycosis – cornea; occurs due to eye trauma; pain and blurring of vision may occur; may lead to blindness ▪ MOT: inoculation of spores on eyes C. Otic infections – external ear infections ▪ Caused by A. niger or A. fumigatus ▪ S/S: pain, decreased hearing, discharge with fluffy green or black growth in the ear canal o Green – A. fumigatus o Black – A. niger D. Allergies ▪ Asthma ▪ Farmer’s lung – hypersensitivity reaction leading to pneumonitis E. Aspergilloma – fungus balls within cavity of old tuberculosis lesion ▪ Seen in people with chronic lung diseases F. Allergic bronchopulmonary aspergillosis – asthmatic symptoms G. Mycotoxicosis – food poisoning as a result of ingestion of toxins ▪ Aspergillus flavus ▪ Aflatoxin – one of the most potent toxins H. Traumatized tissue when spores are inoculated onto the skin Laboratory Diagnosis • Microscopic examination ▪ Demonstration of organism in the tissues (autopsy or biopsy), respiratory samples ▪ Diagnostic features – septate hyphae, dichotomous hyphae (Vshaped branching), spores ▪ Spores or conidia may be uniseriate or biseriate a. Uniseriate – phialides (flask-shaped conidiogenous cells) attach directly to the vesicle ⬥ A. fumigatus K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

b.

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Biseriate – possess structures called metulae ⬥ Metulae - attach directly to vesicle ⬥ Phalides – attach directly to each metula

Culture ▪ Colonies: fluffy, granular, or powdery ▪ Color: 1. A. fumigatus – white to blue green; powdery appearance 2. A. niger – black (1-5 days) 3. A. flavus – yellow to green 4. A. terreus – tan to cinnamon ▪ Culture is used when isolating organisms from tissues ▪ SDA is used ▪ Incubated at RT (since it is a mold) ▪ ID: based on the characteristic spores produced by the species

Treatment • Aspergilloma – surgical excision of infected tissue • Invasive pulmonary and disseminated disease – amphotericin B (preferred regimen) or itraconazole (2nd line regimen)

Zycomycosis • Opportunistic mycoses involving coenocytic fungi belonging to Class Zygomycetes (Zycomycota) ▪ Saprophytic ▪ Aseptate (coenocytic) ▪ Produce sporangiospores ▪ Rapid growers • Infections of the nasal mucosa, palate, lungs, and other vital organs ▪ Abscesses ▪ Ischemia/obstruction • Propensity for vascular dissemination • Causative agents: a. Rhizopus b. Mucor c. Lichtheimia (formerly Absidia) d. Rhizomucor e. Syncenphalastrum f. Cunninghamella • Ubiquitous fungi (found worldwide) • Immunocompromised patients are highly affected

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Minimal pathogenicity among immunocompetent persons

Clinical Manifestations A. Rhinocerebral Mucormycosis ▪ MOT: inhalation; starts as sinusitis ▪ Aka: rhinosinus, sinus orbital or craniofacial zygomycosis ▪ Germination of the sporangiospores in the nasal passage ▪ Invasion of the sinuses, eyes, cranial bones, and brain ▪ Invasion of the hyphae into the blood vessels, causing thrombosis and necrosis = obstruction B.

Thoracic Mucormycosis ▪ Follows inhalation of the sporangiospores with the invasion of the lung parenchyma and vasculature ▪ Causes ischemic necrosis with massive tissue destruction

C.

Cutaneous Mucormycosis ▪ Infection of skin and soft tissues ▪ Spread by hematogenous route following dissemination

Laboratory Diagnosis • Specimens ▪ Skin scrapings from cutaneous lesions ▪ Sputum and needle biopsies from pulmonary lesions ▪ Nasal discharges ▪ Scrapings and aspirates from sinuses in patients with rhinocerebral lesions ▪ Biopsy tissue from patients with gastrointestinal and/or disseminated disease • Direct examination or culture of nasal discharge/tissue ▪ Reveal broad hyphae with uneven thickness, irregular branching at right angles, and sparse septations (coenocytic) ▪ Methods: KOH, stains (H&E, GMS) • Culture – SDA without cycloheximide ▪ Saprophytes are inhibited by cycloheximide Agents A. Rhizopus ▪ Most common cause of mucormycosis ▪ With rhizoids (root-like structures for attachment); unique feature ▪ May also produce brown pigment ▪ Erect, unbranched sporangiophores with rhizoids ▪ Sporangium is usually collapsed K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪ Presence of stolons ▪ Sporangiospores – oval ▪ Columella – ellipsoidal B. Mucor ▪ Erect, branched sporangiophores ▪ Sporangium is intact ▪ No rhizoids ▪ No growth above 37o C C. Rhizomucor ▪ Morphologically intermediate between Rhizopus and Mucor ▪ Rudimentary rhizoids ▪ Branched conidiophores ▪ Grows at temperatures as high as 58o C D. Lichtheimia (formerly Absidia) ▪ Hyphae: broad and ribbon-like ▪ Sporangiophore – slightly branched ▪ Sporangiospores – smooth and ovoid ▪ Presence of internodal rhizoids o Short, thin projections that anchor growing cells to substratum Treatment • Surgical debridement • Rapid administration of amphotericin B • Control of underlying disease

Pneumocystis • Causes Pneumocystis pneumonia or PCP (P. carinii pneumonia) • Etiology: Pneumocystis jiroveci (formerly P. carinii) ▪ Inhabits the lungs; formerly classified as a protozoan ▪ Morphologic forms: a. Trophozoites – thin-walled b. Pre-cyst c. Cyst (oocyst) – infective stage; thick-walled spheres containing 4-8 intracystic bodies

Clinical Manifestations • Causes interstitial plasma cell pneumonia • Infection is asymptomatic among healthy individuals

Immunocompromised (AIDS, premature babies, etc.) – lifethreatening pneumonia ▪ IPCP among AIDS patients S/S: non-productive cough, dyspnea, low grade fever ▪

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Laboratory Diagnosis • Specimens – bronchoalveolar lavage (gold standard), lung biopsy tissue, or induced sputum ▪ Demonstration of oocysts or trophozoites in lung tissues obtained in open lung biopsy o Oocysts – flattened or punched pingpong ballappearance ▪ H&E: widening of alveolar septa with mononuclear cell infiltrates ▪ Foamy exudates within alveolar spaces (honeycombed exudates) o Obstruction in breathing = dyspnea • Stains for detection of cysts and trophozoites ▪ GMS, Giemsa, toluidine blue, calcofluor white ▪ DFA: specific monoclonal antibody Treatment and Prevention • Acute cases: TMP-SMZ + pentamidine isethionate • Prophylaxis: daily TMP-SMZ; aerosolized pentamidine • Improve immune status

Talaromyces marneffei • Causes talaromycosis • Dimorphic fungus; unique among Penicillium species (formerly P. marneffei) • Affected: immunocompetent and immunocompromised patients (HIV patients) • S/S: may have tuberculosis-like symptoms; painless bumps on the face and neck; fever, weight loss; cough; difficulty breathing • Can affect different organs • Chronic infection (weeks to years) • Common in HIV positive patients in Southeast Asia, Southern China, Eastern India • Reservoir host: Bamboo Rat (genus Rhizomys) Laboratory Diagnosis • Specimens – skin lesions, bone marrow, lymph nodes, blood • Direct examination – Giemsa-stained touch smear K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Yeast-like cells (found in tissues) – spherical to ellipsoidal; may have crosswalls Culture: SDA ▪ Yellow pink colonies with diffusible red pigment

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

INTRODUCTION TO VIROLOGY GENERAL CONSIDERATIONS • Structure • Replication • Taxonomy Viruses • Virus = poison • Obligate intracellular organisms ▪ No metabolic systems of their own ▪ Absolutely require living host cells in order to multiply ▪ Highly dependent on the host cell ▪ Very specific (limited number of hosts) • Filterable organisms (contagium vivum fluidum – a contagious fluid) • Virion – it is the complete, fully developed, infectious viral particle a. Genome – nucleic acid core b. Capsid – protein coat o Composed of capsomers/capsomeres/protomers (repeating protein subunits) c. Envelope – additional lipid covering found in some viruses o Labile substance Essential Characteristics 1. Composed of genetic material ▪ Contain a single type of nucleic acid, either DNA or RNA, never both 2. Sizes range from 20-1000 nm ▪ Smallest – Poliovirus ▪ Largest – poxviruses 3. Contain a protein coat (sometimes itself enclosed by an envelope of lipids, proteins, and carbohydrates) that surrounds the nucleic acid 4. Multiply inside living cells by using the synthesizing machinery of the cell 5. Cause the synthesis of specialized structures that can transfer the viral nucleic acid to other cells 6. Host range ▪ Most viruses are able to infect specific types of cells of only one host species ▪ Phages/bacteriophages – viruses that infect bacteria

Viral Genome • Genetic material composed of either DNA or RNA • Viruses contain only one copy of genome (haploid) except retroviruses ▪ Retroviridae – 2 copies of genetic material; diploid • Genetic material may be: a. Single-stranded or double-stranded b. Linear, segmented, or circular A.

B.

DNA Viruses ▪ Usually double-stranded, and linear/circular ▪ Single-stranded members: Parvoviridae, Circoviridae RNA Viruses ▪ Usually single-stranded and segmented ▪ Double-stranded members: Reoviridae, Picobirnaviridae ▪ The genome of RNA viruses may be: a. Sense or plus strand o Viruses that function already as mRNA (i.e., used directly for protein synthesis) b. Antisense or minus strand o Polarity is opposite that of mRNA (i.e., it must be transcribed first) o Cannot be used directly o RNA-dependent RNA polymerase – enzyme used by the virus to transcribe minus RNA to plus RNA to permit translation ▪ Reverse transcriptase (RNA-dependent DNA polymerase) enzyme in retroviruses that reverse transcribes RNA to DNA

Viral Capsid • “Shell” or protein coat that imparts shape and symmetry to the virion • Composed of subunits called capsomeres • Encloses the viral nucleic acid, and protects it from denaturation • Responsible for attachment (tropism), except in enveloped viruses ▪ The capsid has receptors that interact with co-receptors on host cells • Determines specific viral antigenicity, especially in naked viruses (nonenveloped viruses) • Nucleocapsid - term used to describe the viral nucleic acid core enclosed within the protein coat

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Viral Envelope • Antigenic • Surrounds the capsid of some viruses; composed of lipoproteins • Acquired during viral maturation by a budding process through the host cell’s membrane ▪ Derived from the host cell membrane a. DNA viruses - acquire their envelope from the nuclear membrane b. RNA viruses - acquire their envelope from the cytoplasmic membrane • Other structures present on the envelope: 1. Peplomers (spike-like glycoprotein structures) - confer antigenic specificity 2. Matrix - located between the envelope and the capsid; mediate interaction between the envelope and the capsid; enzymatic/ biologic functions, such as inhibition of host-cell transcription • Characteristics of enveloped viruses: 1. Ether-labile/ether-sensitive/ether-susceptible, owing to the hydrophobicity of the envelope 2. Heat-labile 3. More sensitive compared to naked viruses • Factors that damage the envelope: ▪ High and very low temperatures ▪ pH of less than 6 or above 8 ▪ Disinfectants (hydrogen peroxide, phenol) • •

Generally, viruses with helical symmetry are enveloped Mode of transmission: a. Enveloped viruses (susceptible to drying and destruction in the environment) - inhalation, sexual contact, parenteral, direct contact b. Naked viruses - oral-fecal route (more resistant)

Other components of the viral particle: A. Enzymes (e.g., neuraminidase, RNA polymerases, DNA polymerases such as reverse transcriptase) on the envelope surface B. Hemagglutinin (found in influenza viruses) C. Tegument - located between the nucleocapsid and the envelope ▪ Regulatory proteins found in certain viruses ▪ Regulates transcription and translation

STRUCTURAL PATTERNS A. Helical ▪ Shaped like hollow protein cylinders, which may either be rigid or flexible ▪ Nucleic acid and capsid are closely associated ▪ Examples: Tobacco Mosaic Virus, Ebolavirus, Rabies virus ▪ All helical viruses are RNA viruses, but not all RNA viruses have helical symmetry B. Icosahedral ▪ Polyhedral/geometric shape with: o 20 equilateral triangular sides o 12 vertices o 60 identical subunits ▪ Assembled in a cubic manner ▪ Capsids appear spherical when viewed at low power in the electron microscope ▪ Can be crystallized and viewed using radiocrystallography ▪ Example: Adenovirus C. Complex ▪ With symmetry but not purely icosahedral or helical ▪ May possess tails or other structures (e.g., bacteriophages, which posess tail fibers) or have complex, multilayered walls surrounding the nucleic acid (e.g., Poxviruses, such as Vaccinia) Reaction of Viruses to Physical and Chemical Agents • Heat and cold ▪ Heat labile - enveloped viruses ▪ Heat stable - icosahedral viruses ▪ Generally, infectivity is destroyed at 50°-60° C for 30 minutes (except HBV and Polyomaviruses) • Stable with 1 mol/L of salts • pH: generally stable from pH 5-9 • Enveloped viruses - susceptible to ether and detergents • Formalin - inactivates viruses, especially single-stranded viruses • Penetrable by vital dyes • Inactivated by radiation

VIRAL REPLICATION • May be described in two ways: A. Viral Growth Curve B. Viral Growth Cycle K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Viral Growth Curve 1. Virion entering the cell (attachment) 2. Eclipse period (no detectable virion in the host cell) 3. Latent period (patient has no signs/symptoms) 4. Extracellular release of viruses (signs/symptoms appear; cell/tissue destruction)

Poliovirus Rhinovirus Vaccinia virus

Poliovirus receptor (CD 155) ICAM-1 Epidermal growth factor receptor

Penetration • Varied mechanisms for entry • Mechanisms: a. Fusion with the cell membrane (enveloped viruses) b. Endocytosis (enveloped viruses; vacuole formation) c. Translocation (naked viruses; direct penetration by the viral particle) d. Injection of viral material (phages; use of several tail fibers and lysozyme) • Internalizes the virus and also leads to fusion between the infected host cell and additional nearby host cells, forming syncytia Uncoating • Disassembly of capsid to release the genome into the nucleus (DNA viruses) or the cytoplasm (RNA viruses) • Loss of the protein coat, which exposes the nucleic acid

Viral Growth Cycle (Infectious Cycle) 1. Adsorption 2. Penetration 3. Uncoating 4. Macromolecular synthesis (transcription and translation) 5. Assembly and release Adsorption • First step (attachment) • Recognition of a suitable host cell • Specific binding between viral capsid proteins (glycoprotein spikes) and carbohydrate receptors of the host cell Virus EBV (Epstein-Barr virus) HIV HSV 1 Influenza virus Measles virus Rabies virus

Receptor CD 21; C3d, or CR2 CD 4 receptor on T helper cells Fibroblast growth factor receptor Sialic acid CD 46 (SLAM - signalling lymphocytic activation molecule) Acetylcholine receptors, CD 56

Transcription and Translation • Macromolecular synthesis • Replication and expression of genetic material and protein polymers ▪ Early proteins are nonstructural elements (e.g., enzymes) ▪ Late proteins are structural components • Coincides with the eclipse stage • DNA viruses usually replicate in the nucleus, except members of Poxviridae (cytoplasmic replication) ▪ Poxviruses use host cell DNA-dependent RNA polymerase to synthesize mRNA • RNA viruses usually replicate in the cytoplasm, except members of Retroviridae and Orthomyxoviridae ▪ + sense RNA viruses - use host cell RNA polymerase ▪ - sense RNA viruses - use their own RNA-dependent RNA polymerase ▪ Double-stranded RNA viruses - possess their own polymerase ▪ Retroviruses - have + ssRNA that is transcribed into dsDNA by reverse transcriptase (RNA-dependent RNA polymerase) o This copy of DNA is then transcribed into mRNA by host RNA polymerae

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Assembly and Release • Viral maturation (morphogenesis) ▪ Assembly of newly formed viral capsid proteins and genomes ▪ Assembly of new infectious virions • Release of the infectious virions ▪ Mechanisms of release: 1. Lysis of infected cell (naked viruses) 2. Exocytosis of viral particles 3. Budding off (virus acquires an envelope through budding off from the host cell’s membrane) • • • •

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PATHOGENESIS OF VIRAL DISEASES • Viral infection - expression of the viral replicative cycle (partial or complete) in a host cell ▪ Three clinical presentations:

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a. Acute viral infection - early production of infectious virions and rapid onset of disease; evidence of signs and symptoms b. Latent infection - disappearance of signs and symptoms; virus is hidden inside host cells and becomes inactive; virus is present in the host cell in a lysogenic state (inserted into the host genome in a resting state) or as a nuclear/ cytoplasmic episome c. Chronic/persistent infection - long-standing (e.g., HBV); risk factor for carcinogenesis; low levels of virus Clinical disease - presence of overt signs and symptoms Syndrome - specific group of signs and symptoms that are correlated with a disease/infection Tropism - predisposition of a virus towards a specific host/body site ▪ e.g., hepatitis viruses are hepatotropic Viremia - presence of viruses in blood following a local viral infection (often in a mucosal site) ▪ Affects target tissues distant from the primary site of infection ▪ Releases mediators of immune cell functions ▪ Secondary viremia occurs in skin, salivary glands, kidneys, brain tissue, where symptoms manifest Disease disappears when viral replication is stopped by specific antibody and cell-mediated immune mechanisms Viral shedding - expulsion of infectious viral particles from an infected individual; a patient is very infectious when there is a high rate of viral shedding Tissue damage occurs by: ▪ Lysis of infected cells ▪ Immunopathologic mechanisms (cross-reactivity) Latency of viruses in host tissues - characteristic of herpes viruses ▪ During childhood, HSV causes stomatitis. A period of latency following this infection is marked by the disappearance of signs and symptoms. Years later, the virus inside host cells is reactivated by factors such as stress, caffeine, or sunlight and causes cold sores. Autoimmune pathogenesis - occurs well after the acute viral infection has resolved ▪ e.g., Coxsackieviruses resemble receptors expressed by beta-cells of the pancreatic islets. Antibodies produced against the virus crosss-react with the beta-cells, resulting in their destruction and consequent development of DM (molecular mimicry) Oncogenesis (HPV, EBV, HIV, HHV 8, Polymaviruses, HBV, etc.) - oncoviruses (oncogenic viruses) promote transformation or immortalization of host cells K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Effects of Viral Infection on the Host Cell • Cytopathology - cytopathic effects ▪ Visible manifestations of a viral infection ▪ Indication that viruses have already replicated in a host cell ▪ Structural changes in a cell leading to necrosis ▪ Seen in virus-infected cell cultures (diagnostic purposes) ▪ May be seen as: a. Plaque formation b. Formation of multinuclear giant cells c. Cytoplasmic vacuoles d. Inclusion bodies composed of viral particles (e.g., Negri bodies, which are characteristic of rabies infection; eosinophilic cytoplasmic inclusions found in pyramidal neurons and Purkinje cells of the cerebellum) e. Cell degeneration and necrosis • Apoptosis - programmed cell death ▪ Natural mechanisms of the host cell to interrupt further viral replication and to prevent destruction of the host cell by virusinduced necrosis, which results in inflammation and further tissue damage ▪ Defense mechanism of the host cell • Noncytocidal infection - one in which no tissue destruction is evident ▪ No extensive restructuring of host cell ▪ Virions are released by budding • Latent infections ▪ Viral integration into the cell but no viral replication ▪ No disease manifestation; host cell is not damaged ▪ Viral replication may resume several weeks or many years after initial infection ▪ Seen in infections with HSV 1, HSV 2, VZV, CMV, EBV, measles • Immune evasion - virus is not detected by the immune system (may be due to appearance of new antigenic determinants) • Cell fusion - formation of giant syncytia with mulitple nuclei Transmission A. Horizontal ▪ Within a group of individuals a. Direct transmission o Fecal-oral (smear infection) - Enteroviruses o Aerogenic (droplet infection) - Influenza viruses o Intimate contact (mucosa) - Herpes simplex viruses

b. Indirect transmission o Alimentary - Hepatitis A virus o Arthropod vectors - Yellow fever virus o Parenteral - Hepatitis B virus B. Vertical (from infected mother to fetus) Portals of Entry • Mucosa of respiratory and GI tracts • Mechanical inoculation • Skin • Intimate contact

Common Viral Pathogens 1. Cutaneous rash - HSV, VZV, measles, mumps, rubella, HHV 6 2. Meningitis - Coxsackievirus, Echovirus, mumps 3. Keratitis/conjunctivitis - HSV, VZV, adenovirus 4. Parotitis - mumps, parainfluenza 5. Respiratory infections ▪ Adults - rhinovirus, coronavirus, adenovirus, influenza, parainfluenza ▪ Infants/children - same as with adults, plus RSV 6. Myocarditis/pericarditis - Coxsackievirus, Echovirus 7. Hepatitis - HAV, HBV, HCV 8. Infectious mononucleosis - EBV 9. Gastroenteritis ▪ Adults - Norovirus ▪ Infants - Rotavirus, adenovirus 40-41

CLASSIFICATION AND TAXONOMY OF VIRUSES • Bases: ▪ Morphology ▪ Physicochemical properties ▪ Genome ▪ Macromolecules ▪ Antigenic properties ▪ Biological properties • International Committee on Taxonomy of Viruses ▪ Family names end in -viridae (25 families of clinical importance) ▪ Subfamily names end in -virinae (13 subfamilies of clinical importance) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Genus names end in -virus (66 genera of clinical importance) Viral species: group of viruses sharing some genetic information and host (common names are used); Subspecies - designated by a number

Baltimore Classification • Based on genome, strandedness, sense, and method of replication Group I dsDNA

Group II ssDNA

Group III dsRNA (ambisense)

Group IV + sense ssRNA

Group V - sense ssRNA

Picornavirida e

Orthomyxoviridae

Adenoviridae

Group VI Reverse transcribin g RNA

Group VII Reverse transcribing DNA

Retroviridae

Hepadnaviridae

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Arenaviridae Caliciviridae

Herpesviridae

Paramyxoviridae Parvoviridae Reoviridae

Togaviridae

Poxviridae

Bunyaviridae Flaviviridae

Papovaviridae

Rhabdoviridae Coronaviridae Filoviridae Hepeviridae

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LABORATORY DIAGNOSIS • Four methods currently in use: c. Direct detection d. Nucleic acid-based detection e. Isolation of viruses using cell cultures f. Serology • Clinical laboratories provide different levels of service based on their capabilities, resources, and demand • Full service laboratories - viral culture and ID using cell cultures ▪ Offer cell culture, ELISA, and immunofluorescence methods ▪ Equipment required: Laminar flow BSC, fluorescence microscope, inverted bright field microscope, refrigerated centrifuge, refrigerator/freezer, roller drum (holds cell cultures during incubation) • Other laboratories - provide rapid tests specific for different viruses • Viruses that should not be propagated in BSL-1 or BSL-2 laboratories: H5N1, SARS-CoV, hemorrhagic fever viruses, smallpox Specimen Collection and Handling • Specimens should be collected in the early stages of the infection (high viral shedding)

Specimens are collected during the first 3-4 days after the onset of signs and symptoms ▪ The virus may no longer be present as early as 2 days after the appearance of symptoms ▪ Adenovirus and Enterovirus - prolonged shedding in stool ▪ CMV - prolonged shedding in urine Specimens should be collected in strict observation of aseptic technique Specimens are transported and processed immediately, or if not possible, must be processed within 12-24 hours ▪ RSV - more difficult to recover even a few hours after collection Specimens are stored at room temperature ▪ For delays in processing, specimens are stored at 4° C for no more than 5 days ▪ For longer delays and specimen transport, maintain at -70° C for 6 or more days ▪ Specimens should never be stored at -20° C because this temperature facilitates the formation of ice crystals that will disrupt the host cells and result in loss or viral viability Viral transport media - maintain viability and stabilize the virus ▪ Saline or trypticase soy broth may be added to sterile containers to keep tissues moist ▪ Commercial viral transport media are available o Composed of buffered isotonic solution with albumin, gelatin, or serum o Ant ibiot ics are added (penicil l in, vancomycin, streptomycin, amphotericin B) o Examples: 1. M4 2. Universal Transport Media 3. Hank’s Balanced Salt Solution 4. Veal Infusion Broth 5. Leibovitz Emory medium 6. Stuart’s medium 7. Amie’s medium 8. Eagle’s tissue culture 9. Viral Culturettes 10.Virocult ▪ Specimen:transport media (1:2 - 1:5) ▪

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Swabs allowed: dacron, cotton, or rayon, with plastic or aluminum shafts Swabs not allowed: calcium alginate (inhibits viral replication and interferes with nucleic acid amplification tests and-fluorescent-antibody tests), K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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charcoal, and swabs with wooden shafts (contain formalin that inactivates fatty acids in enveloped viruses) Specimens that do not require transport media: ▪ Blood, bone marrow ▪ CSF, amniotic fluid ▪ Urine ▪ Pericardial and pleural fluids Specimens that require transport media: ▪ Respiratory samples ▪ Swabs ▪ Tissue samples For optimal recovery, it is best to sample the infected site directly ▪ Respiratory mucosa secretions - for respiratory infections ▪ Aspirates, surface swabs - for lesions ▪ Stool - intestinal infections ▪ Vesicular skin lesions (VZV, HSV) - unroofing of lesion to make a Tzanck smear/preparation (look for CPEs) In processing specimens, make sure it is accompanied by a request Specimens are processed under a biosafety cabinet/hood

Throat, Nasopharyngeal Swabs or Aspirates • Nasopharyngeal aspirates are preferred over swabs ▪ Throat swabs are acceptable for recovering enteroviruses, adenoviruses, and HSV ▪ Nasopharyngeal swabs/aspirates are preferred for the recovery of Respiratory Syncytial Virus (RSV), Influenza, and Parainfluenza viruses ▪ Nasal specimens - for rhinoviruses • Collection ▪ Throat specimens - rub the inflamed, vesiculated or purulent areas of the posterior pharynx with a dry sterile swab (avoid the mouth, gums, teeth, or buccal mucosa) ▪ Nasopharyngeal secretions - insert a swab with a flexible shaft through the nostril to the nasopharynx or use a bulb syringe with 3-7 mL buffered saline. To collect the aspirate, the saline is squirted by squeezing the bulb and aspirating it back ▪ Emulsify in viral transport media and inoculate • Bacterial contaminants may be removed by concentrating the sample through centrifugation and filtration Bronhcial and Bronchoalveolar Lavage • Washings and lavage fluid collected following bronchoscopy • Excellent for recovery of adenoviruses and influenza viruses

Rectal Swabs and Stool Specimens • Useful for rotavirus, enteric adenoviruses (Serotypes 40 and 41), Norovirus, and enteroviruses • Stool specimens are preferred over rectal swabs • Collection: ▪ Rectal swabs are collected by inserting a swab 3-5 cm into the rectum and rotated to obtain feces ▪ 5-10 mL of freshly voided diarrheic stool for rotavirus and adenovirus ▪ The specimen is placed in viral transport medium and centrifuged at 1000 g for 15 minutes, and inoculate the supernatant Urine • For both molecular and culture methods • For the detection of CMV, mumps, rubella, measles, polyomavorises and adenoviruses • At least 10 mL of first morning urine should be collected by midstream clean-catch • Increased recovery of viruses by: ▪ Addition of 7.5% sodium bicarbonate to neutralize the acidic pH ▪ Multiple collections (2-3) • Filter using 0.2 µm-pore filter to remove bacteria • Inoculate directly if clear ▪ Turbid specimens should be centrifuged at 1000 g for 15 minutes, and the supernatant is used for inoculation Skin and Mucous Membrane Lesions • For the detection of HSV, VZV, and rarely, CMV • There is a low chance of recovery if the lesions of ulcerated or crusted • Tzanck smear preparation: ▪ For detection of VZV and HSV ▪ Skin should be cleansedwith alcohol and allowed to dry for at least 1 minute ▪ Unroof the vesicle or lesion using a sterile scalpel, and carefully remove excess fluid by dabbing with a sterile gauze ▪ Press a clean glass slide against the base of the ulcer to create an impression smear. Send the slide to the lab for fixation and staining ▪ Scrapings and fluid may also be collected from the vesicle and placed in a transport medium ▪ Now widely replaced by DFA Sterile Body Fluids other than Blood (CSF, pericardial and pleural fluids) • For the recovery of enteroviruses, HSV, VZV, influenza virus, or CMV K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• Usually collected aseptically by physicians • Inoculated directly into the culture Blood • Detection and quantification of viremia, especially for CMV, but also for adenoviruses, HSV, VZV, enteroviruses • 5-10 mL of blood is collected and placed in evacuated tubes containing anticoagulants such as: ▪ Heparin - viral recovery from whole blood; may inactivate HIV ▪ EDTA - preferred for collecting plasma for nucleic acid testing ▪ Citrate - used if viruses other than CMV are considered • Separate leukocytes for processing especially for CMV and EBV Bone Marrow • Recovery of viruses that cause aplastic anemia, congenital anemia, chronic red cell aplasia, and hemophagocytic lymphohistiocytosis • Specimens are collected by aspiration and placed in a sterile tube with EDTA • Parvovirus B19 Eye • For viruses causing conjunctival, corneal, or retinal disease • Conjunctival swabs - collected from the lower conjunctiva using a flexible, fine shafted Dacron or Rayon swab moistened with sterile NSS and placed in viral transport medium Tissue Specimens • Usually used for the recovery of viruses that infect the: ▪ Lungs (CMV, influenza, adenovirus, sin nombre virus) ▪ Brain (HSV) ▪ GI tract (CMV) • Collected during surgery • The tissue is minced with sterile scalpel and scissors and gently ground. 20% suspension is prepared in viral transport medium. The suspension is centrifuged at 1000 g for 15 minutes, and the supernatant is used as incoulum • For nucleic acid testing: 25 mg of fresh tissue is minced then treated with proteolytic enzymes ▪ Nucleic acids can also be obtained from formalin-fixed, paraffinembedded tissue Genital Specimens • HSV and HPV • Genital swabs for ulcers; cervical specimens are collected using a swab/ brush Serum Samples • For detection of specific viral antibodies among acute and convalescent patients

Acute specimens - can be collected as soon as possible after the onset of symptoms ▪ Convalescent specimens - collected at least 2-3 weeks after the acute specimens Volume - 3-5 mL collected by venipuncture Serum should be separated from the clot as soon as possible Detection of IgM antibodies should be done prior to freezing to avoid a false-negative result (IgM cold agglutinins bind to RBCs at cold temperatures) ▪

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Virus HSV 1 and HSV 2 CMV Adenovirus Enterovirus Influenza A and B Parainfluenza

Specimens Vesicular lesions, throat swabs, genital lesion swabs Urine, blood, milk, semen, vaginal/cervical secretions, saliva, tears, stool, BAL Nasal, throat, conjunctival and rectal swabs Feces, throat swabs, rectal swabs, CSF, blood, conjunctival swabs, urine Throat swabs, nasal swabs/washings Throat or nasal swavs

DIRECT METHODS OF DETECTION • Microscopy • Detection of Viral Antigens • Nucleic acid detection Microscopy • Large viruses, such as members of the family Poxviridae, are visible under the light microscope • Not as sensitive as culture methods but can offer quick results for rapid therapy • Electron microscopy ▪ Detection of virions ▪ Useful for the detection of non-culturable viruses (e.g., Norwalk, Astrovirus, Calicivirus, Coronavirus) ▪ Not sensitive o Detection limit - viral titer of > 106 to 107/mL Negative staining method employed (viruses are seen as light ▪ structures against a dark background) using stains such as: o Lead o Phosphotungstic acid o Uranyl acetate - most superior stain • Detection of cytopathic effects ▪ Detection of visual changes in virus-infected cells ▪ Smears are stained with Giemsa, Pap’s, or H&E ▪ Presence of cytopathic effects is pathognomonic for the presence of a specific viral infection K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Virus Rabies Yellow Fever Molluscum Contagiosum HSV Poxviruses (Vaccinia,Variola) Adenovirus Measles CMV HPV Enteroviruses Influenza Mumps Parainfluenza RSV Rhinovirus VZV

Cytopathic Effect Inclusion body Inclusion body Inclusion body Inclusion body

Inclusion body Inclusion body

Multinucleated giant cell (syncytia) Inclusion body

Comments Negri bodies Councilman body Lipschultz body or Henderson-Paterson bodies (hyalinized, intracytoplasmic inclusions) Cowdry type A (eosinophilic nuclear inclusions) Rounded, swollen refractile cells. Occasional syncytia, especially with HSV 2. Rapidly involves entire monolayer Guarnieri bodies (B-type inclusions; pinkish cytoplasmic blobs on epithelial tissues) Cowdry type B Rosette type Rounding and aggregation of infected cells in grapelike clusters High affinity for hematoxylin Dawson bodies (SSPE) or Warthin-Finkeldey cells (about 100 nuclei in one cell) Owl-eye inclusions (halo surrounding basophilic nuclear inclusions) Discrete, small foci of rounded cells Koilocytes (virus-infected squamous cells; large nucleus + ground-glass chromatin pattern; wrinkled prune appearance) Cowdry type B (polioviruses) Characteristic refractile angular or tear-shaped CPE; progresses to involve entire monolayer Destructive degeneration with swollen, vacuolated cells CPE usually absent. Occasional syncytia seen CPE usually absent or minimal Syncytia on HEp-2 cells Characteristic rounding refractile cells In PMK, CPE is identical to that produced by enteroviruses Discrete foci of rounded, swollen, refractile cells. Slowly involves entire monolayer Cowdry type A

Antigen Detection • Direct Fluorescent Antibody Testing (DFA) - makes use of virus-specific fluorescein-labelled monoclonal or polyclonal antibody • Indirect immunofluorescence - antigen-capture techniques • Latex agglutination • Enzyme immunoassay - the most popular viral testing method in hospitalbased laboratories • RIA • Immunoperoxidase • ELVIS (enzyme-linked viral inducible system) NUCLEIC ACID DETECTION (Molecular Methods) • Quicker releasing of results • More sensitive • Quantitative • Detects unculturable viruses (Norwalk virus, hepatitis viruses) • Can detect multiple viruses simultaneously (multiplex) • Costly; needs more specialized training

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Methods: PCR, nucleic acid probes, hybridization tests

VIRAL CULTURE • Gold standard (use of cell or organ cultures) • Organ cultures - rarely used (research purposes only) ▪ Useful for the culture of coronaviruses • Use of embryonated eggs - rarely used in diagnostic labs ▪ Once used for orthomyxoviruses (influenza) • Animal inoculation (Coxsackie A viruses require suckling mice for isolation) • Cell cultures - more commonly used ▪ Usually composed of fibroblasts from skin or embryonic lung • Host cells grow into a monolayer on the sides of glass or plastic test tubes ▪ Cells are kept moist and provided with nutrients (Eagle’s minimum essential medium) o Growth medium - initiates growth of cells; rich in serum (10%); prepared with Eagle’s minimum essential medium in Hank’s or Earle’s balanced salt solution; antibiotics added o Maintenance medium - maintains a steady state of metabolism; lower serum concentration (0-2%) ▪ Antibiotics (penicillin, streptomycin, or gentamicin and amphotericin B) can be added to prevent contamination ▪ Main problem - bacterial contamination o Mycoplasma pneumoniae - most common contaminant; fried-egg colonies • Incubation - 35-37° C or 33° C for respiratory viruses ▪ Roller drums (5-7° tilt to allow growth only on one side; 0.5-1 rpm rotation to continually bathe the cells in the medium) • To maintain a physiologic pH (7.2), a bicarbonate buffering system is added • pH indicators (phenol red) is added to the cells to monitor adverse pH changes in the culture ▪ Yellow - acidic ▪ Red - neutral ▪ Purple - basic • Duration of incubation ▪ 1-4 weeks, but usually for 2 weeks Cell Culture • Cell cultures become cell lines if they have been passed or subcultured in vitro • Three categories of cell lines: a. Primary - passed only once or twice K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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o Examples: Human Embryonic Kidney (HEK), Rabbit Kidney (RK; HSV), Primary Monkey Kidney (PMK), Rhesus Monkey Kidney (RMK), African Green Monkey Kidney (AGMK) o Use: sensitive to influenza and parainfluenza viruses, mumps, enteroviruses, and adenoviruses b. Diploid cell lines/Low passage (finite) o Diploid cell lines must have at least 75% of cells with the same karyotype as the normal cells of the tissue o Remain viable for viral growth from 20-50 passages o With increasing passage, the cells become more insensitive to viral infection o Examples: Human diploid fibroblasts such as lung fibroblasts (standard diploid cell line; WI-38 or MRC-5); useful for CMV (does not grow in cell lines other than HDFs) c. Continuous/Heteroploid/Immortal cell lines o Capable of infinite passage o Fused/merged with tumor cells o Cell lines with less than 75% normal cells and more than 25% of the cells having an abnormal karyotype (malignant) o Usually obtained from malignant tissues o Use: sensitive to HSV, VZV, CMV, adenovirus, and rhinovirus o Examples: 1. HeLa cell line (Henrietta Lacks, who died of cervical cancer; her cervical cancer cells were used to make the HeLa cell line) 2. HEp-2 cell line - from cells of human laryngeal carcinoma 3. KB - from cells of human nasopharyngeal carcinoma 4. A-549 - from cells of human lung carcinoma 5. Vero cell line - from African green monkey kidney 6. LLC-MK2 - from rhesus monkey kidney 7. MDCK - Madin-Darby canine kidney epithelial cells Cells are examined for CPEs after incubation (inclusion bodies, rounding, clumping, vacuolization, granulation) CPEs are observed using an inverted light ot phase contrast microscope under LPO

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CPE Grading ▪ Negative - uninfected monolayer ▪ Equivocal - atypical alteration of monolayer involving few cells ▪ 1+ - 1-25% of monolayer exhibit CPE ▪ 2+ - 26-50% of monolayer exhibit CPE ▪ 3+ - 51-75% of monolayer exhibit CPE ▪ 4+ - 76-100% of monolayer exhibit CPE 2+ and above - significant; perform another test to confirm the identity of the virus Other viruses may not exhibit typical CPEs but instead produce: ▪ Syncytia - seen in Parainfluenza viruses 1 and 2 ▪ Hemadsorption - presence of RBC plaques due to adherence of RBCs to the virus o Influenza A and B, Parainfluenza, Mumps ▪ Hemagglutination - due to hemagglutinins o Influenza A and B ▪

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Centrifugation-Enhanced Shell Vial Culture • Rapid modification of conventional cell culture • Cells are grown on round coverslip in a shell vial • Shell vial is inoculated with specimen and centrifuged (to enhance viral absorption of the monolayer) and incubated for 1-2 days or 1-5 days (reduced duration of incubation) ▪ Coverslip is stained and fluorescein-labelled monoclonal or polyclonal antibodies specific for viral antigens are added ▪ Results are visualized using a fluorescence microscope • Once used for Chlamydia • Advantage ▪ Infected cell monolayer is stained for viral antigens right after infection even before development of CPE ▪ For viruses that have longer incubation periods such as CMV and VZV • Disadvantage - only a single type of virus per shell vial SEROLOGY • Most common method, but provides limited information • Measures host response (antibody production), not the virus itself (immunocompromised hosts, such as those infected with HIV, may not mount a measurable immune response) • Usually takes 3-4 weeks before being useful • Results are affected by antibody-producing capacity of hosts K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Antibody levels do not always correlate with the acuteness (activity level) of infection because this is also host-dependent Sample: paired sera (acute and convalescent samples) ▪ A seroconversion or fourfold rise in titer establishes a diagnosis of recent infection IgG and IgM antibodies Serologic tests include: ▪ Complement fixation ▪ ELISA ▪ Western Blot ▪ Indirect immunofluorescence Uses ▪ Diagnosis of nonculturable viruses (e.g., Hepatitis) ▪ Diagnosis of a past (IgG) or acute (IgM) infection from various viral pathogens ▪ Determination of immune status in cases of rubella, VZV, HAV, and HBV ▪ Monitoring of immunosuppressed or transplant patients ▪ Epidemiologic or prevalence studies TORCH panel ▪ Set of tests for the detection of Toxoplasma and others such as Rubella, CMV, and HSV (path


K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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ogens transmitted vertically) Testing is done among pregnant women to screen for pathogens capable of causing congenital defects, miscarriage, and organ defects among infants Serologic testing

ANTIVIRAL DRUGS • Two important concepts: 1. Antiviral drugs attack steps in viral replication and have no effect on latent viruses. Therefore, they are only virustatic, not virucidal. 2. Most are nucleotide analogues. They are taken up by DNA polymerase or reverse transcriptase to inhibit them, causing termination of protein synthesis. Agent Idoxuridine Adenine arabinoside Acyclovir Gancyclovir Ribavirin Azidothymidine (AZT) Dideoxycytidine (ddC) Amantadine Foscarnet Saquinavir Inteferons (alpha, beta, gamma)

Mechanism of Action Halogenated pyrimidine is phosphorylated in vivo by thymidine kinase and replaces thymidine in DNA to block DNA synthesis Also known as vidarabine. A purine. Phosphorylated in vivo on the unnatural sugar and arabinose. Blocks viral DNA polymerase Inhibits thymidine kinase and DNA polymerase. Useful against HSV Similar to acyclovir but useful for CMV Nucleoside analogue that inhibits guanosine-5-phosphate. MOA not fully understood Also known as zidovudine. Inhibits viral reverse transcriptase and stops DNA elongation Behaves exactly like AZT Inhibits uncoating of Influenza A and may interfere with ion channels in the viral envelope Not a nucleoside analogue. It is a phosphporus-bearing compound that inhibits both DNA and reverse transcriptase First protease inhibitor to be approved for HIV treatment Cytokines that promote a cellular antiviral state. IFN-alpha has been used to treat Hepatitis B and C

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

DNA VIRUSES GENERAL CONSIDERATIONS • DNA viruses comprise six families: 1. Herpesviridae 2. Hepadnaviridae 3. Adenoviridae 4. Poxviridae 5. Papovaviridae (now separated into Polyomaviridae and Papillomaviridae) 6. Parvoviridae 7. Circoviridae • All have linear genome, except for the following which have circular genome: ▪ Papovaviridae ▪ Hepadnaviridae • All have double-stranded genome, except for the following which have single-stranded genome: ▪ Parvoviridae ▪ Circoviridae • All are naked, except for the following which are enveloped: ▪ Herpesviridae ▪ Hepadnaviridae ▪ Poxviridae • All are icosahedral in structure, except for Poxviridae (complex) • All replicate in the nucleus, except Poxviridae (replicate in the cytoplasm) • Oncogenic DNA viruses (tumor-associated): ▪ Human Papilloma Virus – cervical cancer ▪ EBV – Burkitt lymphoma ▪ HBV – liver carcinoma ▪ HHV8 – Kaposi sarcoma (AIDS patients) ▪ Polyomavirus ▪ SV40 virus • Smallest DNA virus – Parvoviridae • Largest DNA virus – Poxviridae

HERPESVIRIDAE • Most diverse (8 human herpes viruses known) • herpes- “to creep” General Characteristics • Linear double-stranded DNA genome • Enveloped, icosahedral, and possess an amorphous tegument ▪ Tegument – 20 different regulatory proteins for viral replication; enter the host cell upon fusion of the envelope and cell membrane and initiate the replication cycle • Develop a latent state during infection, wherein S/S disappear ▪ Activated from latency by fever, emotional stress, caffeine, exposure to UV light, or axonal injury • Similar appearances under the EM, but different effects • Zoonotic herpes viruses do not infect humans, except Herpes B virus (causes a fatal encephalitis) • Divided into three subfamilies: A. α-Herpesvirinae B. γ-Herpesvirinae C. β-Herpesvirinae α-Herpesvirinae Type

Genus

Virus

Target Cell

Site of Latency

MOT

Simplexvirus

HSV1

Mucoepithelial

Neuron (trigeminal ganglion)

Close contact; respiratory secretions and saliva

Simplexvirus

HSV2

Mucoepithelial

Neuron (sacral ganglion)

Close contact; sexual contact, perinatal infection

Varicellovirus

VZV (chickenpox)

Mucoepithelial

Neuron (dorsal root ganglion)

Respiratory and close contact

HHV1

HHV2

HHV3

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Present with vesicular rashes (fluid-filled lesions) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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γ-Herpesvirinae Type

Genus

HHV4

Lymphocryptovirus

HHV8

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Rhadinovirus

Virus

Target Cell

EBV

B cell and epithelial cells

Kaposi’s sarcomaassociated herpesvirus

Lymphocytes and other cells

Site of Latency

MOT

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B cells

Close contact; kissing

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B cells

Close contact; sexual contact, saliva

γ-Herpesvirinae viruses target lymphocytes and are associated with cancer β-Herpesvirinae Type

HHV5

HHV6

HHV7

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Genus

Virus

Target Cell

Site of Latency

MOT

Cytomegalovirus

CMV

Monocytes, lymphocytes, epithelial cells

Monocyte, lymphocyte

Saliva, urine, breast milk

Roseolovirus

Roseolovirus, Herpes Lymphotropic Virus

T-cells

T cells

Respiratory and close contact

Roseolovirus

Pityriasis Rosea

T cells and ?

T cells

Multinucleated giant cells are seen in: ▪ HHV 1-3, and HHV5

HERPES SIMPLEX VIRUSES • Very common • Most infected patients are asymptomatic • Two categories of infection: a. Primary – first or initial infection (acquired in childhood) b. Recurrent – reappearance after several years due to physiologic stress; results from reactivation of the latent virus • Mode of Transmission – direct contact with the infected tissue lesions (oral, genital, mucosal); causes direct cytopathology; usual IP: 2-11 days ▪ HSV 1- generally transmitted orally (saliva); lesions are above the waist

HSV 2 – transmitted by sexual contact; genital herpes; lesions below the waist; more readily reactivated compared to HSV-1 In certain circumstances, disease manifestations may be reversed (i.e., due to oral sex): HSV 1 causes genital herpes and vice versa To differentiate the two, use molecular methods or monoclonal antibodies

People at high risk: ▪ Children (HSV 1) ▪ Sexually-active individuals (HSV 2) ▪ Medical professionals in contact with oral and genital secretions for infection of fingers (herpetic whitlow – lesions on fingers of health professionals) ▪ Immunocompromised and neonates = at risk for disseminating disease Pathogenesis • Latency – virus evades the immune system by hiding in neurons a. HSV 1 – trigeminal ganglion b. HSV 2 – lumbar and sacral ganglion • Reactivation – occurs during periods of stress and immunosuppression • Causes an infection that persists throughout life • Asymptomatic shedding of the virus may occur • Cytolytic Clinical Manifestations (HSV 1) • Oral herpes – usually caused but not exclusive to HSV 1 ▪ Intraoral mucosal vesicle or ulceration ▪ Affected areas: buccal mucosa, posterior pharynx, gingival and palatal mucosa ▪ Fever blisters – gingivostomatitis; 1o HSV 1 infection o Primary herpes gingivostomatitis – most common HSV 1 infection in children • Herpes Labialis/Facialis/Febrilis – recurrent HSV infection; cold sores due to reactivated disease; occurs at the border of the lips at the junction of the oral mucosa and skin • Other diseases: a. Keratoconjunctivitis/ocular herpes – corneal ulcers leading to blindness; swelling of the eyelids associated with vesicles o Dendritic ulcers which are branching o 2nd most common cause of blindness o HSV - most common cause of corneal infection o Involves only the superficial epithelial layer (heals completely with treatment) •

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

b.

c. d. e.

Sporadic encephalitis – necrotic lesion in one temporal lobe; leading cause of sporadic encephalitis o Caused by HSV-2 in neonates o Caused by HSV-1 in older children and adults o HSV-1 - most common cause of viral CNS infection (viral neurotropic spread through the olfactory bulb) Herpetic whitlow – among medical professionals Herpetic gladiatorum – wrestlers; neck and trunk lesions Disseminated infections – neonates and immunocompromised

Clinical Manifestations (HSV 2) • Genital herpes (herpes genitalis) – caused by HSV 2 in 80-90% of cases ▪ Painful vesicular lesions; recurrence is less severe ▪ Females – vesicles appear on the vaginal/labial mucosa, or both; cervix and vulva may also be involved; urethra ▪ Males – shaft, glans, and prepuce of the penis; urethra ▪ Double the risk for HIV infection if there is current HSV 2 infection •

•

Neonatal (congenital) herpes – acquired by neonates from infected mothers in utero, during birth (intrapartum), or after birth (postnatally) ▪ Severe cases = may lead to encephalitis ▪ Less severe cases = vesicles ▪ Asymptomatic ▪ More severe if HSV-2 is involved ▪ Higher risk if the mother has a primary infection Erythema multiforme ▪ Caused by both HSV 1 and HSV 2; may also be caused by other agents ▪ Rash – target or bull’s eye lesions ▪ Areas – trunk, hands, feet

Diagnosis • Direct microscopic exam ▪ Preparation of Giemsa-stained Tzanck smear on herpetic vesicles to visualize cytopathic effects (multinucleated giant cells and Cowdry Type A inclusion bodies) • Cell culture – gold standard ▪ CPEs can be seen within 1-2 days ▪ Samples must be inoculated into cell culture within 1 hour after collection • Serology – EIA, ICT • Brain biopsy for encephalitis (CSF not recommended)

• •

Molecular methods (DNA detection in CSF using PCR) - more sensitive than culture and antigen detection NAT – nucleic acid testing

Treatment – acyclovir, valacyclovir, famciclovir VARICELLA ZOSTER VIRUS • Morphologically and structurally similar to other herpes viruses but differs in antigenicity • Disease – varicella/chickenpox (primary infection) and zoster/shingles (recurrent form) • MOT – droplet inhalation or direct contact with the lesions • Occurs in epidemic proportions • 90% of the general adult population has contracted VZV in childhood • Site of infection – conjunctiva or mucosa of the upper respiratory tract, then travels to the lymph nodes ▪ Primary viremia - infected T cells from the lymph nodes enter the blood ▪ Secondary viremia - infected T cells invade the liver, spleen, etc., then the viruses infect skin cells Clinical Manifestations • Incubation period – 2-3 weeks • Fever, malaise, pruritic rash and vesicular lesions ▪ Lesions begin on head and trunk, then spread to limbs (centrifugal spread) ▪ Lesions crust within 1-2 days, but do not resolve for approximately 3 weeks • Rashes (different stages [in order of appearance]): a. Papule: erythematous b. Vesicles: fluid-filled c. Pustules: pus-filled d. Crusts: dried ▪ ▪ ▪ ▪

Rashes develop asynchronously (i.e., several stages may be seen on the skin at the same time) Very infectious before appearance of the rash and during convalescence Appearance of vesicles = dewdrop on a rose petal As opposed to smallpox, whose lesions are found in more abundance on the skin of the extremities, chickenpox lesions are found on the head and trunk K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

• • •

Complications: ▪ Teens and adults – severe form of disease with potential pneumonia ▪ Immunocompromised and newborns – life-threatening pneumonia, encephalitis ▪ Reye’s syndrome – encephalopathy and liver degeneration due to high levels of ammonia o Develops in individuals who were given aspirin Immunity = life-long Latency – dorsal root or cranial nerve ganglia Reactivation of VZV = shingles/herpes zoster ▪ Factors for reactivation – stress and immunosuppression among adults and the elderly ▪ Shingles – also known as zoster; reactivated form of VZV infection ▪ Presentation: o Rash followed by vesicular lesions in a unilateral dermatome pattern (naka-hilera; painful since lesions develop near nerve endings) o Prolonged disabling pain that can remain for months even after the lesions have disappeared (post-zoster neuralgia or postherpetic neuralgia) o Occurs many years after the primary infection o From the dorsal or cranial nerve ganglia, virus travels to the skin ▪ The formation of neutralizing antibodies against the anti-VZV antibody triggers shingles

Diagnosis • Tzanck smear – presumptive diagnosis ▪ CPE: multinucleated giant cells (swollen epidermal cells containing intracytoplasmic viral infclusion) • Cell culture – definitive diagnosis ▪ Small clusters of ovoid cells in fibroid cells in MRC-5, HF, A549 ▪ Use human embryonic lung or Vero cells (CPEs are seen after 3-7 days) • Antigen detection ▪ Addition of specific fluorescent-labeled antibodies • Molecular methods EPSTEIN-BARR VIRUS • Disease: infectious mononucleosis (also known as mono, kissing disease, Pfeiffer’s disease, glandular fever)

•

MOT: close oral contact (kissing), sharing of personal items such as toothbrushes, etc.

Pathogenesis • Virus is found in saliva and infects oral epithelial cells ▪ Spreading occurs to B cells (receptor – CD21 and complement C3d receptor) ▪ Once the virus infects the B cell, the infected B cell is transformed ▪ Infected B cells produce many copies of the EBV DNA and is passed on to the B cell progeny • Virus is cytolytic (infected B cells are destroyed by T cells) • Some viruses hide in the B cells (latency) • Immunocompromised states = virus may be reactivated ▪ Uncontrolled growth of B cells, leading to Burkitt’s lymphoma (malignant; common in the immunocompromised and in African children) ▪ Also associated with nasopharyngeal carcinoma, as well as Hodgkin’s and non-Hodgkin’s lymphomas ▪ Transplant recipients - high risk of lymphoma Clinical Manifestations • Highly affected: young adults (infected through social contact); children may be asymptomatic or have mild symptoms • S/S: fever, chills, sweats, headache and very painful pharyngitis; lymphadenopathy may also occur (tonsillitis), hepatomegaly and splenomegaly ▪ Hairy leukoplakia – hair-like structure on the lateral portion of the tongue; seen in AIDS patients • Production of anti-i antibody (IgM; associated with cold agglutinin disease) • Malignancy – Burkitt’s lymphoma, nasopharyngeal carcinoma ▪ Reactivation of the EBV ▪ High risk: immunocompromised and elderly Laboratory Diagnosis • Blood picture = lymphocytosis with atypical lymphocytes (Downey cells) ▪ Downey cells – variant/atypical T lymphocytes; ballerina skirt appearance • Culture – using human B lymphocytes • EBNA test – detect the presence of EBV nuclear antigen • Heterophile antibody test ▪ Monospot test – slide test ▪ Paul-Bunnell heterophile antibody test

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

EBV-specific antibodies produced: a. Anti-VCA (viral capsid antigen) antibody IgM o Seen in the early course of infection; disappears in about 4 weeks o Indicates current infection b. Anti-VCA IgG – appears in acute stages and persists for life c. Anti-EA IgG - appears in the acute phase; indicates current or recent infection o No longer detected after 6 months d. Anti-EA/D (Ab to early Ag, diffuse) – appear in acute stage and indicates current or recent infection; no longer detected after 6 months o Patients with nasopharyngeal carcinoma have high levels of Anti-EA/D IgG and IgA e. Anti-EA/R (Ab to early Ag, restricted) – appears in acute phase; can persist up to 2 years o Patients with Burkitt’s lymphoma have high levels of AntiEA/R IgG f. Anti-EBNA (EBV nuclear antigen) – appears 1 month after infection; levels peak in 6-12 months

•

PB

Anti-VCAIgM

Anti-VCAIgG

Anti-EAIgG

Anti-EBNA

Interpretati on

-

-

-

-

-

No previous exposure

+

+

+

+/-

-

Acute infection

+/-

+/-

+

+/-

+

Recent infection

-

-

+

-

+

Past infection

CYTOMEGALOVIRUS (HHV 5) • Infected cells exhibit cytomegaly (enlargement) • Largest genetic content of all herpes viruses • Associated diseases: cytomegalic inclusion disease, Mono-like disease (mistaken for IM), diseases affecting immunocompromised and transplant patients • MOT – acquired orally, sexually, from blood transfusions, in utero, at birth, by nursing ▪ Shed in urine, saliva, tears, stool, and breast milk • Infection is very common but most are asymptomatic ▪ At risk: pregnant women, immunocompromised Pathogenesis • Target host cell – epithelial cells, lymphocytes • Site of latency – monocytes, T cells, macrophages • People at risk: neonates, sexually active individuals, immunosuppressed, organ transplant patients (life-threatening systemic disease) • The virus is shed constantly in the urine during the course of the infection Clinical Manifestations • Most are asymptomatic ▪ 80% of adults are infected with no apparent symptoms • Cytomegalic inclusion disease – affects neonates ▪ Occurs when CMV crosses the placenta ▪ Causes petechiae, hepatosplenomegaly, mental retardation, microcephaly, chorioretinitis deafness, seizures, and other birth defects ▪ May occur if pregnant mother becomes infected ▪ If the disease occurs during the 1st trimester = interference with brain development • CMV is a leading cause of congenital defects in the US ▪ Skin findings: purpuric papules and nodules • Among immunocompromised and transplant patients ▪ Pneumonia, pneumonitis, hepatitis • AIDS patients: intractable colitis with diarrhea, retinitis ▪ CMV is the most common cause of AIDS-related retinitis • CMV mononucleosis/heterophile negative mononucleosis ▪ Mono-like symptoms and hepatitis • Reactivation – in immunocompromised patients (e.g., AIDS and transplant patients) ▪ Retinitis, pneumonia, disseminated infections, colitis, and death ▪ Asymptomatic shedding of the virus in urine

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Laboratory Diagnosis • Specimens - normally sterile body fluids (buffy coat and other internal body fluids • Cell culture ▪ CPE: Owl’s eye inclusions in the nucleus o Specimens can be cultured using urine or respiratory secretions ▪ Shell vial technique – diagnosis within 24-72 hours ▪ Isolated only by using human diploid fibroblasts (e.g., human embryonic lung or human foreskin fibroblasts) ▪ Takes up to 3 weeks for the CPE to appear • Serology: IgM/IgG ▪ Fluorescent antibody techniques ▪ Antigen detection (detection of pp65) o pp65 – marker of antigenemia; secreted by leukocytes; 65-kDa lower-matrix phosphoprotein o Useful to assess the efficacy of treatment • Molecular methods - determine viral load • For congenital infections, specimens must be collected within the first 2 weeks of life (urine) Preventive and Control Measures • No vaccine • Antiviral drugs for AIDS patients • Refrain from sexual contact • Screening blood and organ donors (pregnant, immunocompromised, organ recipients) • Treatment: ganciclovir and foscarnet HUMAN HERPESVIRUS 6 • Lymphotrophic virus that targets T and B cells ▪ Viral receptor: CD46 ▪ Site of latency: T and B cells, monocytes • MOT: inhalation of aerosols, close contact ▪ Viral shedding in saliva • Causes roseola infantum or exanthema subitum, or 6th disease ▪ Rashes ▪ Targets infants • Two serologically similar variants: a. Variant A – causes disease in the immunocompromised b. Variant B – causes the disease (roseola infantum, exanthem subitum, sixth disease) o Common among children aged 6 months-2 years

•

•

• •

Roseola infantum or exanthem subitum ▪ Febrile illness with mild constitutional symptoms lasting 3-5 days ▪ After defervescence = pink macular or maculopapular rash appears on the trunk ▪ Lasts hours to days ▪ Infants are protected by maternal antibodies until about 6 months of age ▪ Seizures Other associated diseases: ▪ Progressive multifocal leukoencephalopathy (PML) o Targets oligodendrocytes ▪ Multiple sclerosis – autoimmune disease targeting the myelin sheath of neuronal axons Reactivation of latent infection occurs in the immunocompromised and may result in pneumonia Laboratory diagnosis ▪ Cell culture – use of lymphocytes ▪ Serology ▪ PCR and viral load testing - most sensitive ▪ Clinical findings

HUMAN HERPESVIRUS 7 • May also cause exanthem subitum but less common (causes roseola) • Also shed in saliva • Target: T lymphocytes (viral receptor: CD4) • Site of latency: T cells • Quite similar to HHV 6, but antibodies to HHV-6 does not protect against HHV-7 • Older children (6 years and above) usually become seropositive • Latency may occur • Diagnosis: culture (peripheral/cord blood lymphocytes), PCR, serology HUMAN HERPESVIRUS 8 • Also known as Kaposi sarcoma-associated herpesvirus (tumor-causing) • Target: B cell • Site of latency: B cell • MOT: sexual contact; transplantation ▪ Seen among HIV-infected patients ▪ MSMs are more prone to infection • The virus is able to cause malignant transformation by inactivating the retinoblastoma tumor suppressor protein with the use of the nuclear antigen K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• •

•

•

Also plays a role in the development of primary effusion lymphomas and multicentric Castleman disease Kaposi’s sarcoma ▪ Malignancy common among AIDS patients ▪ Affects the vascular endothelium ▪ Systemic disease with cutaneous lesions (patches) seen in the lining of the nose, mouth, throat Diagnosis ▪ Immunohistochemistry ▪ PCR ▪ In-situ hybridization for HHV-8-infected tissue ▪ Cannot be cultured B virus – affects monkeys; in humans = myelitis and encephalomyelitis

HEPADNAVIRIDAE • Circular, dsDNA • Enveloped, icosahedral capsid • Dane particle – complete viral particle • Causes hepatitis B or serum hepatitis • Humans are the only source • Heat-stable; retains infectivity in drying blood and other body fluids for severe days • Fatal disease is most likely to occur in people co-infected with hepatitis D • MOT ▪ Parenteral (blood, intimate contact, needles, perinatal) ▪ Median incubation period: 3 months • Terms a. HBV – the agent for hepatitis B b. HBsAg – surface antigen; most reliable marker for identifying HBV infection c. HBeAg – envelope antigen; indicates viral replication high infectivity and a chronic carrier state d. HBcAg – core antigen e. Anti-HBs – indicates convalescence or past infection; immunity f. Anti-HBe – suggests presence of HBV; g. Anti-HBc – indicates past infection; lifelong marker

HBsAg

Anti-HBs

HBeAg

Anti-HBe

Anti-HBc

-

-

-

-

-

+

-

+/-

-

-

Early acute HB

+

-

+

-

+

Acute or chronic HB

+

-

-

+

+

Chronic HBV carrier state

-

-

-

+

+

-

+

-

+

+

Recovery from HB with immunity

-

+

-

-

-

Distant HBV infection of HB vacciine

•

•

Interpretat ion No (or very early) exposure to HBV

Early recovery phase from acute HB

Clinical syndromes ▪ Acute hepatitis ▪ Fulminant hepatitis – severe acute hepatitis with rapid liver destruction ▪ Chronic hepatitis ▪ Primary hepatocellular carcinoma HBV DNA in serum (PCR) - best indication of active viral replication and a high state of infectivity ▪ Also used to predict the patient’s response to treatment

ADENOVIRIDAE • First recovered from adenoid tissue • Linear, dsDNA • Naked, icosahedral capsid • Presence of protruding fibers from each of the 12 vertices of the capsid ▪ Orbiting satellite appearance under EM ▪ Presence of hexons and pentons o Hexons – 6 subunits o Pentons – 5 subunits; where the vertex is located Vertex fibers – for attachment; hemagglutinin ▪ K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

• • • •

•

• • •

•

~ 52 serotypes ▪ Serotyping is based on protein coat ▪ Most common: 1 to 8, 11, 21, 35, 37, and 40 Seven subgenera: A to G Genus: Mastadenovirus Very stable (remains viable for weeks at variable temperatures in surfaces and in liquid solutions) MOT: aerosols, fomites, oral-fecal, personal contact ▪ Shed in secretions from the eyes and respiratory tract Associated diseases: a. Respiratory tract infections (colds, tonsillitis, pharyngitis, croup, fatal pneumonia): serotypes 1, 2, 3, 5, 7 b. Gastroenteritis (diarrhea in young children): serotypes 40 and 41 (enteric adenoviruses); less vomiting and fever than with rotavirus infection c. ARD (3, 4, 7, 14) – severe acute respiratory distress seen among military recruits d. Epidemic keratoconjunctivitis (8, 19, 37) – sore eyes; adults; transmitted through fomites e. Swimming pool conjunctivitis (3, 7) o Prevented by chlorination f. Acute hemorrhagic cystitis g. Pharyngoconjunctival fever Latency occurs in the lymphoid system (tonsils, adenoids, Peyer’s patches) People at risk: people at daycare centers, military training personnel, camps, swimming clubs Diagnosis ▪ EIA, IFA, Antigen Detection Kits, culture, shell vial, serology ▪ CPE – grape-like appearance o Inclusions have high affinity for hematoxylin o A-549, HEp-2, HeLa ▪ Serotyping: by serum neutralization or hemagglutination inhibition Vaccine – formerly available for serotypes 4 and 7 given to military recruits

POXVIRIDAE • Largest (200-400 nm) and most complex of all viruses • Linear, dsDNA • Brick-shaped complex virus with a disk/dumbbell-shaped core ▪ Multilayered and possesses a lipoprotein envelope ▪ Presence of lateral bodies • Replicates in the host cell cytoplasm

• • •

•

▪ Possesses DNA-dependent RNA polymerase Virus is assembled in its inclusion bodies called Guarnieri bodies (B-type inclusions) Changes the cellular structure of epidermal cells to make it seem like there are pocks on the skin MOT – inhalation (respiratory infection) of aerosol droplets, face-to-face contact and spread mainly in the lymphatics; direct contact; through fomites Viruses of medical importance: a. Smallpox/Variola virus (Genus Orthopoxvirus) b. Vaccinia virus (Genus Orthopoxvirus) – used for vaccination against smallpox c. Molluscum contagiosum virus (Genus Molluscipoxvirus) – common in HIV patients ▪

Cowpox – source of the vaccine made by Edward Jenner

VARIOLA • Smallpox (bulutong) • Acute contagious disease of the reticuloendothelial, vascular endothelial, and epithelial cells • Synchronous progressive rash with fever ▪ Result of viral replication in the skin, followed by damage caused by T cytotoxic cells attacking the infected cells • Now eradicated/extinct • MOT – inhalation of aerosol droplets ▪ Initial site of replication and infection = upper RT ▪ From the upper RT, virus travels to the lymph node, blood, internal organs, blood (secondary viremia), skin = rashes • Rashes are found in the face and the extremities ▪ Macule (small, round changes in skin color) to papule (slightly elevated with no fluid) to vesicle (containing a bubble of fluid) to pustule (with purulent material consisting of necrotic inflammatory cells) ▪ Rashes are deep-seated = resulting scars are disfiguring Chickenpox

Smallpox

Mode of Evolution

Lesions appear in crops

Lesions progress from stage to stage synchronously

Time of Evolution

Rapid

Relatively slow

Lesions

Superficial; oval or totally irregular

Deep set; tend to be circular and regular K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Vesicles

•

•

• •

•

Unilocular; scarring light and superficial

Multilocular (coalescing); scarring severe and deep

Two categories: ▪ Variola major – severe form; more deadly; causes more disfigurement ▪ Variola minor – Alastrim; milder form of variola Reasons why smallpox has been eradicated (WHO declared the virus eradicated in 1980) ▪ Disease is exclusive to humans; no animal reservoir ▪ 1 serotype (univalent vaccine) ▪ Disease always presents with visible pustules ▪ Identification of sources and contagion ▪ Availability of a stable, inexpensive and easily administered vaccine (Vaccinia) ▪ Presence of scar indicates successful vaccination ▪ Political commitment ▪ Successful WHO worldwide campaign Eradicated by the use of live attenuated vaccine (Vaccinia) administered intradermally using a bifurcated/forked needle Prevention and diagnosis ▪ Eradicated by the use of live attenuated vaccine (Vaccinia) ▪ Diagnosis: cell culture, growing of virus in chick embryo, viral antigen detection by IF Treatment - cidofovir or brincidofovir

VACCINIA • Used to vaccinate against smallpox • Disease: localized exanthema through epithelial cell infection • MOT: sexually transmitted; skin contact • 1st reservoir host – rabbits • Lesions are larger than smallpox with necrotic centers MOLLUSCUM CONTAGIOSUM • Disease of children and young adults • Disease: molluscum contagiosum/water warts • MOT: direct skin contact, intimate contact, indirect contact • Manifestation: presence of small papules on the skin or mucous membrane ▪ Cup-shaped crater; dome-shaped papules which are flesh or pinkcolored • Seen in immunocompromised and AIDS patients • Treatment: removal of the lesion

MONKEYPOX VIRUS • First described in primates (first case of human infection in 1970) • Introduced into the US by rodents imported from Africa (Gambian rats) • Vesicular, pustular febrile illness very similar to smallpox, but is less severe ORF VIRUS • Transmitted from sheep to humans through direct contact • Causes single or multiple nodules (usually on the hands) • Autoinoculation on the eye poses serious consequences

PAPILLOMAVIRIDAE • Naked, circular dsDNA • Genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapapillomavirus, Nupapapillomavirus ▪ Species: Human Papilloma Virus ▪ Most clinically significant HPVs belong to Alphapapillomavirus ▪ More than 100 serotypes (more than 40 are sexually-transmitted) ▪ Some serotypes are oncogenic (cancer-causing) • MOT: sexual contact, use of shared objects, prenatal • Tropism for epithelial cells of either cutaneous or mucosal types • Site of latency: epithelial cells ▪ Initial site – stratum basale ▪ Site of replication – stratum corneum • Diseases ▪ Warts (verrucose or papillomas) – finger-like benign lesions ▪ Carcinomas of the cervix, penis, anus, oropharynx • Clinical manifestations ▪ HPV serotypes have a predilection to infect certain parts of the body ▪ Usually subclinical, no apparent symptoms • Cutaneous warts – found in the skin (HPV 1-4) a. Verrucae vulgaris (2-4) – common wart; seen in hands, feet, knees; no associated malignancy b. Plantar warts (1) – found on the hands, soles of feet; inward growth of warts; painful; no associated malignancy c. Flat and macular warts (verrucae planae) – found on the skin of the forehead, arms, and face o 5, 8, 9, 12, 14, 15, 17, 19-25, 36-38 o > 30% of patients with epidermodysplasia verruciformis with types 5, 8, 14, 17, and 20 develop malignancy

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

d.

•

•

•

•

Butcher’s warts (7) – occupational hazards to butchers; warts are seen in the hands e. Genital warts (6, 11) – venereal warts or condyloma acuminata o May appear as flat or keratotic warts o Easily transmitted to sexual partners o Cervical HPV lesions - consist of flat areas of dysplasia; rinsing with 5% acetic acid turns the lesions white and makes them more visible o Most prevalent sexually-transmitted viral disease Carcinomas – cervix, penis, anus, oropharynx ▪ Certain serotypes may pose a high risk of association to malignancy ▪ Serotypes 16 and 18 – cervical cancer ▪ Serotype 16 – oropharyngeal cancer ▪ Serotype 18 - adenocarcinoma ▪ Mechanism of carcinogenesis: o E6 and E7 genes = encode proteins that inactivate the p53 and retinoblastoma proteins respectively ▪ Immunosuppressed patients and those with AIDS ▪ HIV-(+) women are at higher risk of developing cervical cancer Diagnosis ▪ Cannot be grown in cell culture ▪ Clinical picture ▪ Cytology sections ▪ PCR ▪ Pap’s smear o Look for koilocytes (cells with perinuclear clearing with an increase in density of the surrounding cytoplasmic rim) = hallmark of infection o Koilocyte nuclei have a wrinkled prune appearance Prevention: vaccination ▪ Gardasil quadrivalent vaccine against serotypes 6, 11, 16, and 18 o Indicated for females 9-26 years old o May also be used for males ▪ Ceravix - protects against serotypes 16 and 18 ▪ Gardasil-9 - further protects against several lower-risk HPV types known to be associated with cervical and most HPV-induced oral cancers Treatment - removal of warts by surgery, cryotherapy, and laser

POLYOMAVIRIDAE • Small, circular, dsDNA • Icosahedral, naked

• • • • •

Tumor-causing MOT – direct contact with respiratory secretions Site of latency – kidney and B cells Infection usually occurs during childhood High risk: immunocompromised

JC Virus • John Cunningham virus • Disease: progressive multifocal leukoencephalopathy ▪ Demyelinating disease ▪ Affects oligodendrocytes and astrocytes ▪ AIDS patients • Specimen - brain tissue BK Virus • Disease: hemorrhagic cystitis • Seen in bone marrow transplant patients • Seen in immunocompromised renal transplant patients • First isolate from a renal transplant patient with the initials B.K. • Specimen - urine MC Virus • Causes Merkel cell carcinoma SV40 (Simian Polyomavirus 40) • Can induce tumors in animals and associated with human tumors • Seen in patients vaccinated for polio (contaminated Salk polio vaccine)

PARVOVIRIDAE • Smallest DNA virus (Lt. “parvus” = small) • Naked, icosahedral, ssDNA • Spherical under EM • Surface proteins – VP1 and VP2 ▪ VP1 – minor surface protein ▪ VP2 – major surface protein • MOT – respiratory and oral secretions such as saliva, sputum, or nasal mucus • Target sites – erythroid progenitor cells, adult bone marrow, fetal liver cells ▪ Targets hematopoietic tissues = causes cell lysis ▪ Crosses the placenta • Genera K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

a.

•

Dependovirus – virus dependent upon helper viruses such as Herpesviruses and Adenoviruses for replication o Defective virus (i.e., cannot replicate in the absence of helper viruses) b. Erythrovirus – does not require helper cells to replicate o Only medically important virus = Parvovirus B19 (associated with aplastic anemia) Parvovirus B19 ▪ Single human pathogen among the Parvoviridae ▪ Named after the serum sample (number 19 of panel B) in which the initial viral isolate was observed by EM ▪ Viral receptor – P antigen found in RBCs, megakaryocytes, etc. ▪ Clinical presentation: o 5th disease (erythema infectiosum): rashes in children o Aplastic crises (aplastic anemia) – at risk are children with chronic anemia; pancytopenia o Hydrops fetalis – HDN; virus can cross the placenta and infect the fetus o Arthritis – females; immune complex deposition to the joints (small joints of feet and hand bilaterally) o Chronic B19 infection – affected: HIV patients, chemotherapy patients, transplant patients ♦ Chronic anemia, leukopenia, thrombocytopenia ▪ Erythema infectiosum o Also known as 5th disease o Causes a biphasic illness among humans i. Initial/prodromal phase – marked fever, malaise, myalgia, chills; viremia; erythroblast destruction ii. Second phase – maculopapular rash, arthralgia, arthritis; virus disappears in the blood; diagnose by antibody detection ♦ Rash – slapped cheek appearance; occurs as a result of immune complex deposition in skin capillaries ♦ Associated with immune complex deposition in the capillaries of the skin o Third stage – begins as the rash starts to fade with areas of central clearing; this leaves a reticulated or lacy pattern of erythema which can last several weeks ♦ Most common complication – joint involvement, ranging from mild arthralgias to overt arthritis

•

Diagnosis ▪ Enzyme immunoassay (IgG and IgM) ▪ PCR (blood or amniotic fluid)

•

Human Bocavirus (HBoV) - upper and lower respiratory tract infections ▪ Present in co-infections with RSV and human metapneumovirus (hMPV) ▪ Shed in high quantities in stool

ORIGINAL SIX EXENTHEMATOUS DISEASE 1.

Measles

4. Dukes

2.

Scarlet Fever

5. Erythema infectiosum

3.

Rubella

6. Exanthem subitum

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

RNA VIRUSES GENERAL CONSIDERATIONS • All are single-stranded, except: ▪ Reoviridae ▪ Picobimaviridae • All possess a helical symmetry, except: ▪ Hepeviridae ▪ Caliciviridae ▪ Picornaviridae ▪ Flaviviridae ▪ Togaviridae ▪ Reoviridae ▪ Retroviridae ▪ Astroviridae • All are enveloped, except: ▪ Picornaviridae ▪ Caliciviridae ▪ Hepeviridae ▪ Reoviridae ▪ Astroviridae • All replicate in the cytoplasm, except: ▪ Retroviridae ▪ Orthomyxoviridae • RNA viruses with segmented genome: ▪ Retroviridae ▪ Arenaviridae ▪ Bunyaviridae ▪ Orthomyxoviridae ▪ Reoviridae • Largest RNA virus: Paramyxoviridae • Smallest RNA virus: Picornaviridae • Arboviruses (arthropod-borne) ▪ Bunyaviridae ▪ Flaviviridae: Dengue, Yellow fever ▪ Togaviridae: Chikungunya ▪ Reoviridae: tick-borne

FAMILY PICORNAVIRIDAE • Most diverse • +-sense ssRNA • Naked, icosahedral • Capsid: made up of 4 proteins (VP1-VP4) ▪ VP1-VP3: surface proteins ▪ VP4: internal ▪ VP1 and VP3 – major antibody-binding sites • VPg (viral protein genome-linked): inside capsid ▪ Serves as a primer for genome replication • Made of twelve (12) genera ▪ Important members: a. Enterovirus: Poliovirus, Coxsackievirus, Echovirus ♦ Includes Rhinovirus (common cold) b. Parechovirus c. Hepatovirus (Hepatitis A) d. Kobuvirus (Aicihi virus) e. Aphthovirus (causes foot-and-mouth disease in livestock) f. Cardiovirus (targets the hearts and brains of rodents) Enterovirus

Rhinovirus

Mode of Transmission

Aerosol inhalation Oral-fecal Fomites

Aerosol inhalation Contact with secretions Fomites

Site of Infection

GI/alimentary tract (initial), other parts of the body POE – mouth

Upper RT (nose, throat)

Yes Initial site of replication: GALT (gut-associated lymphoid tissue)

No (localizes in the upper RT only) Colds worsen only due to secondary bacterial infection

Preferred Temperature for Replication

37o C

33o C

Other Properties

Acid-stable (pH 3 to pH 9) Resistant to detergents, mild sewage treatment, and heat

Dissemination

Acid-labile

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

ENTEROVIRUS • Targets: a. Intestinal epithelial cells b. Lymphoid tissue • Wide range of clinical manifestations ▪ Fever of unknown origin, aseptic meningitis, paralysis, sepsis-like illness, myopericarditis, pleurodynia, conjunctivitis, exanthema, pharyngitis, and pneumonia • Disease mechanisms: ▪ Lymphoid tissue: site of initial viral replication ▪ Viremia – presence of the virus in blood; dissemination to different organs (CNS, muscles, heart, liver) via the bloodstream ▪ Most patients are asymptomatic or have mild symptoms (immunocompetent patients) o In healthy patients: blocking antibodies formed when virus is replicating in the GALT block the further spread of the virus ▪ Some may have more clinical manifestations depending on the patient’s immune status ▪ Target: extremes of ages (very old and very young) ▪ Patients develop immunity even in the absence of symptoms • Members: a. Poliovirus b. Coxsackievirus c. Echovirus d. Enterovirus 70 e. Enterovirus 71 Poliovirus • Causative agent of poliomyelitis (infantile paralysis) ▪ Flaccid type of asymmetric paralysis o Only ½ of the body is affected o Sensory function is intact ▪ Only humans are affected; no animal reservoirs • Gk. polio (gray) and myelon (matter/spinal cord) • Serotypes: 1, 2, and 3 ▪ Serotype 1 – most commonly implicated in paralysis • MOT: oral-fecal • Virus is rapidly inactivated by UV light, heat, formalin, and chlorine ▪ Infections are usually seen in individuals with poor hygiene Pathogenesis • Initial site of infection – oropharynx, lymphoid tissues and small intestine

The virus is present in the pharynx and stool before the onset of illness Hematologic spread towards the lymphatics and CNS Virus spreads along the nerve fibers, eventually causing nervous tissue destruction ▪ Virus destroys motor neurons located in the: 1. Anterior horn (spinal cord) 2. Brain stem Cause flaccid paralysis or bulbar poliomyelitis ▪ Bulbar poliomyelitis – occurs when the brainstem and medulla are affected o More deadly o Arrest of vital functions (respiratory arrest due to paralysis of diaphragm) Viral receptor – CD155 (Poliovirus receptor [Pvr]) ▪

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Clinical Manifestations A. Inapparent/Asymptomatic ▪ Seen in most cases (90%) ▪ No clinical symptoms; infections are limited to the oropharynx and gut ▪ No paralysis (virus is limited to lymphoid tissue due to blocking antibodies) B. Abortive ▪ Minor illness ▪ Most common clinical form ▪ Non-specific febrile illness (fever, headache, malaise, sore throat, vomiting) occurs within 3-4 days of exposure ▪ Spontaneous recovery ▪ 5% are affected C. Non-paralytic (aseptic meningitis) ▪ Occurs in 1%-2% of patients with infection ▪ Virus progresses into the CNS and meninges ▪ Back pain, muscle spasms (stiffness of neck, back, and legs) + symptoms of the minor illness ▪ Spontaneous recovery (self-limiting) D. Paralytic ▪ Major illness affecting < 1% of the individuals infected with the virus ▪ Appears 1-7 days after the minor illness has subsided (biphasic illness) ▪ Hematologic spread to the CNS (affects the motor cortex) ▪ May lead to: K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

E.

o Flaccid paralysis o Bulbar poliomyelitis (respiratory paralysis) o Meningomyeloencephalitis ▪ Degree of paralysis depends on the extent of nerve damage Post-polio syndrome (progressive post-poliomyelitis muscle atrophy) ▪ Sequalae of polio infection ▪ Occurs many years after the initial infection ▪ Deterioration of the muscles affected by the poliovirus due to loss of nerves (denervation atrophy) o Results in a ragdoll appearance ▪ May result from recrudescence of paralysis ▪ Cause is still unknown

Prevention A. Passive immunization ▪ Administration of antibodies ▪ No immunologic memory B. Vaccination – administer the antigen to trigger an immune response (with immunologic memory) Salk Vaccine

Sabin Vaccine

Formalin-killed/inactivated vaccine

Antigen

eIPV (enhanced polio vaccine) Trivalent Inactivated Polio Vaccine (protection from all serotypes)

Live attenuated poliovirus

4 (2 months, 4 months, 6-18 months, 4-6 years) Doses

3 (given at 6 weeks of age) Vaccine may also be given before the onset of paralysis

Route of Administration

Advantages

Intramuscular

Oral

Effective Stable May be administered to immunodeficient patients (no possibility of wild-type polio)

Gut immunity (stimulates production of IgA, aside from IgG and IgM) Effective; longer immunity Used in high-transmission countries

No gut immunity (no IgA production) Disadvantages

Booster needed

May revert to wild-type polio (cannot be given among immunodeficient patients) Vaccine should be refrigerated Immunization may be impaired by other Enteroviruses (concurrent Enterovirus infection may nullify the effects of the vaccine)

Diagnosis • Use of cell cultures to isolate the virus • Specimens – CSF, stool, oropharynx secretions • Molecular methods • Serology: IFA, ELISA Treatment – no treatment available Coxsackievirus • Two major groups based on murine studies (effects of the virus on rodents): a. Coxsackie A – rapid death of rodents + myositis + paralysis b. Coxsackie B – not fatal to rodents; less severe • MOT – fecal-oral route; respiratory aerosols • High transmission rates during the summer months • Primary site of replication – oropharynx and GI tract • Familial (easily transmitted within the household) Coxsackievirus A – 24 serotypes • Affected sites – skin and mucous membranes • Disease manifestations: ▪ Myositis (skeletal muscles of newborn); may lead to flaccid paralysis ▪ Herpangina – vesicular pharyngitis; painful eruption of vesicle in muscle and throat; oropharynx ▪ Hand, foot, and mouth disease (CAV serotypes 5, 10, 16) o Children affected present with: malaise, headache, abdominal pain o Appearance of maculopapular rashes ▪ Aseptic meningitis – serotypes 7, 9 ▪ Colds K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Coxsackievirus B • Affected organs – heart, pleura, pancreas, liver, CNS • Diseases: ▪ Pleurodynia (Bornholm disease or Devil’s grip) o Painful inflammation of the internal muscles (diaphragm) o Severe thoracic pain when breathing ▪ Pericarditis – Coxsackie B virus is the most common cause of viral heart disease, presenting with pericarditis ▪ Myocarditis ▪ Spastic paralysis ▪ Aseptic meningitis (all types) ▪ Diabetes (CBV serotype 4) – targets the pancreas (β-cell destruction) = mimics type 1 DM Diagnosis • Cell cultures • Serology (serum neutralization tests) Treatment and Prevention • No treatment and vaccine available Echovirus (enteric, cytopathic, human orphan virus) • No formal classification when discovered • Target: children • Disease: infantile diarrhea, aseptic meningitis, upper RT, febrile illness with or without rash • Echovirus 9 – outbreaks of aseptic meningitis Enterovirus 70 – main cause of acute hemorrhagic conjunctivitis Enterovirus 71 – leading cause of viral CNS disease (meningitis, encephalitis, paralysis), HFMD PARECHOVIRUS • Parechovirus A ▪ Causes summer diarrhea ▪ Respiratory infections, meningitis, neonatal sepsis • Parechovirus B – Ljungan virus ▪ Zoonotic; may cause intrauterine fetal death and type 1 DM HEPATOVIRUS • Hepatitis A (infectious hepatitis) • Also known as Enterovirus 72 • MOT: oral-fecal (ingestion of contaminated food and water)

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▪ Street food (fecally-contaminated) Disease: epidemic or infectious hepatitis Pathogenesis: ▪ Virus replicates in the GI and spreads to the liver ▪ Virus is shed in large amounts of feces ▪ Incubation period: 1 month ▪ Children: most frequently affected ▪ Manifestations: fever, anorexia, nausea, vomiting and jaundice; pale stool, dark urine Diagnosis: IgM anti-HAV and IgG anti-HAV; elevated liver enzymes, jaundice

RHINOVIRUS • Common cold • 113 serotypes • MOT: direct contact (person-to-person; respiratory droplets) ▪ Indirect contact • Target: nose and throat; does not cause generalized infections ▪ S/S: rhinorrhea and coryza • Symptoms worsen if there is bacterial superinfection • Symptoms cannot be differentiated from other respiratory viruses • Viral receptor: ICAM-1 (expressed on epithelial fibroblasts and lymphoblastoid cells) • Unable to replicate in the GIT (acid labile) • Diagnosis: ▪ Cell culture (CPEs) ▪ Demonstration of acid lability • Vaccination is not feasible (different serotypes every year)

FAMILY ARENAVIRIDAE • Gk. arena = sand • Under EM: appear sandy and granular ▪ Due to ribosomes of the infected host cell • ssRNA, ambisense • Segmented; helical symmetry; enveloped ▪ Possesses T-shaped or club-shaped spikes/peplomers in the envelope • Two groups: A. Old World – LCM (lymphocytic choriomeningitis virus), and Lassa virus (more severe), Lassa-like viruses (Mopeia, Mabala, Ippy)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

B.

New World – Tacaribe complex virus (Tacaribe, Guanarito, Junin, and Machupo viruses), Amapari, Cupixi, Parana, Latino, Pichinde, Tamiami, Flexal, Sabia, Oliveros, whitewater Arroyo, etc. o Junin - first arena virus found to cause hemorrhagic fever (Argentine hemorrhagic fever) Common in Africa

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Pathogenesis • MOT: inhalation of rodent excrements, direct contact with infected rodents • Zoonotic (rodents) ▪ Rodents – natural hosts (rodent-borne infection); shed the virus in urine, feces, and saliva; do not die from the infection ▪ Humans – dead-end hosts • Cause hemorrhagic fevers

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Clinical Manifestations • LCM – Lymphocytic Choriomeningitis Virus ▪ Natural host – Mus musculus (house mouse) ▪ Flu-like illness that might lead to aseptic meningitis • Lassa Fever – usually asymptomatic ▪ Some patients develop fever, headache, pharyngitis, myalgia, diarrhea, and vomiting ▪ Hemorrhage and CNS involvement may occur; deafness is a common complication ▪ Pantropic – hemorrhage occurs in all internal organs; high mortality ▪ Natural host – Mastomys natalensis (multimammate rat); once infected, it sheds the virus all its life ▪ Deadly in pregnant women ▪ Can be transmitted from person to person through throat secretions or through sexual contact ▪ May be treated with ribavirin Diagnosis • Isolation of virus from blood and liver (post-mortem) • Highly infectious • Cell cultures, serology, molecular methods (RT-PCR) • Biosafety Levels ▪ LCM – BSL 3 ▪ Lassa Virus – BSL 4

FAMILY CALICIVIRIDAE

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Icosahedral, naked +-sense ssRNA VPg – viral protein at the 5’ end of the genetic material Structure has 32 calyx-like (cup-shaped) concavities giving the virus a Star of David appearance of the capsid MOT: fecal-oral route ▪ Low infectious dose (< 100 virions) Affects both humans and animals ▪ In humans: gastroenteritis and diarrhea ▪ In cats: respiratory tract diseases ▪ In swine: vesicular disease ▪ In rabbits: hemorrhagic disease Five genera: a. Sapovirus – Sapporo Virus (human calicivirus), genogroups I, II, IV, V b. Norovirus – Norwalk Virus (human calicivirus), genogroups I, II, IV c. Nebovirus – Bovine enteric viruses d. Lagovirus – Rabbit Hemorrhagic Disease Virus e. Vesivirus – Feline Calicivirus

Norwalk Virus/Norovirus/Small Rounded Structured Virus • Most important cause of epidemic viral gastroenteritis among adults • From Norwalk, Ohio • Causes Winter Vomiting Disease • MOT: food borne, waterborne, person-to-person transmission • IP: 1-2 days • Symptoms: nausea, low-grade fever, diarrhea, vomiting, dehydration • Diagnosis: ▪ Stool - best specimen; collected within 48-72 hours after onset ▪ Cannot be cultured ▪ Direct methods (EM of stool material) ▪ Molecular methods (PCR) o RT-PCR - most common assay used to detect noroviruses • Implicated as a cause of gastroenteritis in cruise ships (spreads easily), as well as in resorts, schools, nursing homes, and families • Immunity is short-lived • Cannot be cultured; detected using RT-PCR Sapporo Virus • Causes gastroenteritis among infants and toddlers (less commonly in school-age children) • Cup-shaped morphology • Prevention: K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪ ▪ ▪ ▪ ▪

Careful disposal of stools Effective handwashing Careful processing of food Purification of drinking water and swimming pool water No vaccine is available

FAMILY HEPEVIRIDAE • Genus Orthohepevirus ▪ Hepatitis E virus ▪ Formerly under Caliciviridae • MOT: oral-fecal • Similar to Hepatitis A; no chronic state • Common in Asia, Central America, and Africa • High mortality among pregnant women (3rd trimester)

FAMILY ASTROVIRIDAE • ssRNA • Non-segmented, naked, icosahedral • Star-like surface appearance ▪ Spikes: 5 or 6 pointed spikes • Very small, size similar to poliovirus • MOT: oral-fecal route • Genus: Mamastrovirus • Disease manifestation: ▪ Gastroenteritis (osmotic/watery diarrhea) among the elderly and the young

FAMILY CORONAVIRIDAE • Enveloped, helical viruses ▪ Source of envelope – endoplasmic reticulum membrane • ssRNA • Possess the longest sense RNA genome (25-32 kilobases in size) • Under EM: appear as solar crown • Possess club-shaped/pearl-shaped/petal-shaped projections ▪ Peplomers – for viral entry; hemagglutinins • Infect a variety of hosts such as humans, and other vertebrates (dogs, cats, rodents, bats, poultry) ▪ Causes respiratory diseases in humans

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▪ Diarrhea in infants Genera that infect humans: ▪ Torovirus, Alphacoronavirus, and Betacoronavirus o SARS-CoV – belongs to Betacoronavirus Disease manifestations: ▪ Colds, rhinitis, pediatric disorders ▪ Novel diseases (SARS-CoV, MERS-CoV) MOT: person-to-person via direct contact, aerosol, or droplets ▪ 2nd week onwards - period of maximum infectivity and highest viral loads in the upper airways

SARS-CoV: Severe Acute Respiratory Syndrome Coronavirus • Caused by a novel coronavirus (SARS-CoV) • Viral pneumonia that affects the lower RT ▪ High-grade fever, pneumonia, acute respiratory distress syndrome • Mean IP: 5 days • Emerged in Guandong, China and HK in 2002 and emerged rapidly to 26 countries • Worldwide pandemic (infected at least 8000 people) • Deadly especially in immunocompromised • Target: ▪ Target receptor – angiotensin-converting enzyme 2 (ACE 2) in epithelial cells of the respiratory tract • Reservoir – horseshoe bat or palm civet cat MERS-CoV: Middle Eastern Respiratory Syndrome Coronavirus • First reported in Saudi Arabia in 2012 and subsequent reports in the Arabian peninsula • Severe respiratory illness with symptoms of fever, cough, shortness of breath • Mortality rate: 50% (there have been 852 cases and 301 deaths) • At risk: elderly, immunocompromised, patients with chronic illnesses Diagnosis • Culture (Vero Cell Line); difficult to culture (extremely fragile), CPE: rapid cell rounding, refractivity, and detachment • Serology: ELISA, IFAT • Molecular: RT-PCR (most common diagnostic approach) • EM • Process using BSL 3 or higher (cell cultures) Treatment • Supportive K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• • FAMILY FILOVIRIDAE • Filamentous and thread-like • The most pathogenic among the hemorrhagic fever viruses • Thread-like or filamentous RNA viruses • Pleomorphic (exhibit branching); appear as a circle or U or 6 • Enveloped, helical virus with protruding peplomers • Two genera: a. Marburgvirus – Lake Victoria Marburgvirus hemorrhagic fever; shepherd’s hook morphology b. Ebolavirus – Ebolavirus Zaire, Sudan, Reston, Tai Forest stains o Discovered in the Democratic Republic of Congo o Zaire strain = most severe o Identity of strains depends on area • Both genera cause: ▪ Hemorrhagic fever (rare occurrence and unknown reservoirs but cause infections with high mortality rates) • MOT: direct contact with monkeys and other infected persons, nosocomial transmission, sexual contact ▪ Close contact with body secretions Lake Victoria Marburgvirus • Hemorrhagic fever contracted from African green monkeys • Fever, maculopapular rash, nausea, vomiting, diarrhea • Bleeding from nose, gums, and GI tract occur at the latter part; ▪ Hemorrhage of vital organs (liver) leading to death ▪ Myocarditis, kidney damage, mental status changes • MOT: nosocomial, casual contact, sexual contact • Diagnosis: PCR, immunohistochemistry, IgM-capture ELISA • Treatment: replacement of blood and clotting factors Ebolavirus • Ebola Zaire – more virulent • Ebola Sudan • Ebola Reston – contact with monkeys; less severe; no development of disease ▪ Reston, Virginia (monkeys from the Philippines) • Symptoms – fever, chills, myalgia, anorexia, sore throat, abdominal pain, diarrhea, vomiting, bleeding • Virulence factor – glycoproteins, proteins that inhibit the induction of IFN ▪ Peplomers target the endothelial cells = hemorrhage

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Probable natural host of Ebolavirus – fruit bats of the Pteropodidae family Diagnosis: ▪ PCR ▪ IF ▪ ELISA ▪ Culture ▪ EM Most recent and largest outbreak: ▪ West Africa Vaccines ▪ ChAd3-ZEBOV - finished Phase I of clinical trials ▪ VSV-EBOV - finished Phase I, now in Phase II and Phase III ▪ Heterologous prime-boost (Johnson and Johnson) - two-dose vaccination process using AD26-EBOV and MVA-EBOV

FAMILY PARAMYXOVIRIDAE • Largest RNA viruses • Non-segmented, minus sense ssRNA • Helical nucleocapsid; pleomorphic • Envelope contains surface glycoproteins (peplomers) – virulence factors a. Hemagglutinin + neuraminidase – merged into 1 peplomer; for attachment b. Fusion – separate peplomer; mediates membrane fusion (promote syncytia formation) + hemolysin • Two subfamilies: a. Paramyxovirinae 1. Paramyxovirus – Parainfluenza virus 1 and 3; RT infections 2. Rubulavirus – Parainfluenza virus 2 and 4; mumps 3. Morbillivirus – measles (Rubeola); only HA and F (no NA) 4. Nonclassified – Nipah and Hendra virus; zoonotic ♦ Nipah – horses; SE Asia ♦ Hendra – pigs; Australia b.

Pneumovirinae 1. Pneumovirus – RSV (respiratory syncytial virus); common in children; only F protein 2. Metapneumovirus – recently discovered

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Virulence factors: Virus

HA

NA

F

Measles

+

-

+

Mumps

+

+

+

RSV

-

-

+

Parainfluenza

+

+

+

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Acute illness characterized by fever, malaise, anorexia and followed by unilateral or bilateral swelling of the parotid gland (parotitis) and other organs o Parotitis is always present Highly affected: children aged 5-9 years Only one serotype: infection or vaccination provides lifelong immunity MOT: close contact with saliva (virus is shed in saliva) Other organs affected: ▪ Testis – orchitis (men who are beyond puberty; may lead to sterility; uncommon) ▪ Pancreas ▪ Ovaries Diagnosis ▪ Specimen – saliva/urine, swabs from the Stensen duct, pharynx, CSF ▪ Direct methods, culture (Vero or LLC-MK2), shell vial ▪ Serology (hemagglutination-inhibition tests, CF detecting the antibody against soluble antigen) ▪ Molecular methods ▪ Mumps skin test – determine previous infection; determine if CMI is competent ▪

MOT – respiratory droplets (droplet infections), direct contact, respiratory secretions, zoonotic transmission (Hendra and Nipah viruses) Affected: most infections are seen in children Target area: initial infection in the RT; some may become generalized in cases of measles ▪ Most members cause RT-localized infections, except measles, which may cause systemic infection

Parainfluenza Virus • Clinical manifestations: ▪ Common cold syndrome (cold-like symptoms) ▪ Bronchitis ▪ Croup (laryngotracheobronchitis): barking sound (seal-like) ▪ Bronchiolitis (PIV 3): in children < 2 years old ▪ Pneumonia • Common in children • All manifestations are respiratory (non-systemic) • Otitis media – usual complication in children • Serotypes: ▪ PIV 1 – croup (children) ▪ PIV 3 – bronchiolitis and pneumonia (infants) ▪ PIV 4 – mild upper respiratory disease • Diagnosis: ▪ Specimens – aspirates and nasopharyngeal washes o Nasopharyngeal swabs ▪ Cell culture (PMK or LLC-MK2 cells) – look for syncytia ▪ Serology (hemadsorption, IF or EIA) – detect antigens ▪ Direct methods: IF Mumps Virus • Disease – mumps (beke)

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Measles Virus • Disease: measles (Rubeola; tigdas) – highly contagious disease of children • S/S: ▪ High fever ▪ Coryza (runny nose) ▪ Koplik’s spots – pathognomonic for measles o Lesions on oral mucosa which consists of irregular red spots with a bluish white speck in the center o Found in the buccal area ▪ Maculopapular rashes – due to T-cell response to virus-infected endothelium lining the capillaries • MOT: aerosol droplets • Very infective during the prodromal period (before appearance of rash with fever, malaise, etc.) • Pathogenesis ▪ Initial site – respiratory tract epithelium, going to the RES (multiplication), blood, skin (rashes) • Common complications: ▪ Otitis media, diarrhea K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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Severe complications: ▪ Bronchopneumonia (leading cause of death among measlesinfected children (< 5 years) ▪ Encephalitis (leading to deafness and mental retardation) Long-term complication: subacute sclerosing panencephalitis (SSPE) ▪ Occurs 7-10 years after acquiring the infection ▪ Mental changes, seizures, then death ▪ Seen in people infected with measles before they were 2 ▪ Defective measles virus that persists in the brain and acts as a slow virus ▪ CNS manifestations: personality, behavior, memory changes, myoclonic jerks, spasticity, and blindness ▪ Death: within 6-12 months Measles in pregnant women – risk for stillbirth Deadlier than Rubella Risk factors for severe measles: ▪ Children: malnourishment, vitamin A deficiency ▪ Pregnant, immunocompromised Diagnosis: ▪ Specimen: nasopharynx and urine ▪ Tests: culture (PMK); look for characteristic CPE (WarthinFinkeldey Giant Cells) and spindle-shaped cells, serology (serum neutralization, EIA, or IF) Prevention: vaccination (live, attenuated vaccine) ▪ MMR or AMV (attenuated measles vaccine) ▪ 2 doses: 15 months, 4-6 years, subcutaneously ▪ Booster is recommended

Respiratory Syncytial Virus (RSV) • Most common virus isolated from infants • 2 serotypes: A and B • Common cause of viral pneumonia and bronchiolitis among children 5 years and below • Other diseases: croup, cold, bronchitis, lower RT infections among adults and the elderly • Infections do not confer complete immunity (infections occur throughout life) ▪ Recurring infection ▪ Previous infections only confer partial immunity • MOT: respiratory droplets; direct contact; entry through nose and eyes • Diagnosis: ▪ DFA or EIA using nasopharyngeal swabs and washes

Cultures are difficult; culture in HEp2 (syncytia formation), PMK, or human diploid fetal cells ▪ Rapid antigen detection kits Treatment - ribavirin, palivizumab ▪

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Metapneumovirus • First described in 2001 • Occurs most often in children • Wide range of respiratory illness from mild upper RT symptoms to severe lower RT disease • Symptoms are similar to RSV • Detected by PCR using clinical samples from children with respiratory illness Nipah and Hendra Viruses • Zoonotic; endemic to Southeast Asia and Australia ▪ Nipah – Southeast Asia ▪ Hendra – Australia • Cause encephalitis and severe interstitial pneumonia • High mortality (40%)

FAMILY ORTHOMYXOVIRIDAE • ssRNA, segmented viruses that cause the flu (trangkaso) • Enveloped, icosahedral capsid • Ortho – true (to differentiate from Paramyxoviridae) • Myxo – mucus (ability to attach to mucoproteins on the cell surface) • Genus: Influenza viruses • Two separate surface peplomers: hemagglutinin and neuraminidase Hemagglutinin

Neuraminidase

80% of peplomers For attachment and entry to host cell Ability to agglutinate RBCs Determines virulence Major antigen against which neutralizing antibodies are directed 16 types of HA of Influenza A (H1 to H16; H1-H3 most common) Rod-shaped peplomer

20% of peplomers (Mushroom-like spikes) Destroys neuraminic acid (sialic acid), a component of the specific cell receptor for these viruses (viral entry is allowed when sialic acid is removed) Main function: release of the new virus from the cells; cleaves budding viruses from infected cells Determines the subtype of Influenza A virus isolates 9 NA types of Influenza A (N1-N9; N1 and N2 most common)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

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▪ HA and NA are essential for serotyping (e.g., H1N1, H5N1) ▪ No F protein Genome: 7-8 segments of ssRNA Matrix proteins – anchors the HA and NA glycoproteins; found in the envelope ▪ Influenza A – 2 types of M protein a. M1 protein – structural integrity (maintain the integrity of the capsid) b. M2 protein – forms an ion channel; for viral uncoating MOT: inhalation of small aerosol droplets released by talking, breathing, or coughing Virus prefers cool, less humid atmospheres (e.g., winter heating season) ▪ Flu is more common during the winter ▪ Usually self-limiting Complications in very young and very old Variant viruses (H3N2v) - viruses that infect humans that usually circulate in animals

Genera A. Influenza A: flu pandemics ▪ Its tendency to cause pandemics lies in its ability to undergo both: 1. Antigenic drift – slow genetic change; mutations ♦ Minor antigenic change; seen as point mutations in the genetic material during replication ♦ Changes in HA and NA ♦ Slower occurrence leading to new viral strains 2. Antigenic shift – abrupt or sudden change in genome ♦ Gene reassortment between 2 influenza viruses ♦ Reassortment of HA and NA antigens to create a novel flu virus ♦ Associated only with Influenza A Antigenic drift and shift are mechanisms employed by Influenza A to produce new viral strains ▪ Infects both humans and animals Influenza B: outbreaks/epidemics ▪ Infects humans only (no animal reservoir) ▪ Only undergoes antigenic drift Influenza C: mild respiratory illness ▪ Infects humans only ▪ Undergoes antigenic drift ▪ Lacks the gene for neuraminidase o

B.

C.

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Basis of difference: ▪ Group specific antigen: viral nucleoproteins and matrix proteins ▪ Type specific antigen: HA and NA

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Mechanisms of antigenic shift: a. Genetic reassortment of non-human and human influenza viruses, producing a virus with new HA and NA b. Direct infection of humans by Flu virus of other animals (pigs or birds) without genetic reassortment c. Non-human flu virus is passed from one type of animal (e.g., birds) through an intermediate host (pig) to humans o Animals (pigs) act as mixing vessels when they are exposed to different strains = produce new strains At risk for Flu illness: ▪ Seronegative individuals ▪ Adults ▪ School-aged children At risk of hospitalization due to Flu: ▪ Children less than 2 years old ▪ Persons with certain medical conditions (diabetes, heart disease, lung disease or immunocompromising conditions) ▪ Pregnant women ▪ Persons 65 years old and above

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History of Flu Pandemics • Three major Flu pandemics in the 20th century: 1. Spanish Flu (1918-1919) – caused by A(H1N1) o Killed 50-100 million people 2. Asian Flu (1957-1958) – caused by A(H2N2) o More than 1 million dead worldwide 3. Hong Kong Flu (1968-1969) – caused by A(H3N2) o More than 1 million dead worldwide • Recent pandemics: 1. A(H5N1) – Bird/avian Flu o Hong Kong, 1998 o Sporadic human infections are far more severe with high mortality; longer incubation period 2. A(H1N1) – Swine Flu o Similar to seasonal flu o 2009 • Dominant strains of Influenza A since 1977: ▪ H1N1: swine flu K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪

H3N2

Pathogenesis • Virus attacks the upper RT (ciliated epithelial cells; causes necrosis and sloughing) • Severity of the disease depends on the immune status, presence or absence of underlying disease of the infected patient • Disease is more common in the winter months • Clinical syndrome: ▪ IP: 1-4 days ▪ Prodrome: malaise, headache ▪ Prodromal stage followed by abrupt onset of fever, myalgia and usually nonproductive cough, coryza (rhinitis) ▪ Acute illness lasts for 1 week but malaise and cough may persist for 2 weeks ▪ Diarrhea – seen in influenza caused by pandemic strains (AH5N1) ▪ S/S: o Headache, fever, extreme tiredness, joint aches, vomiting, coughing, runny or stuffy nose, sore throat, aches • Complications: ▪ Secondary bacterial complications and fatal pneumonia ▪ Exacerbation of pre-existing conditions ▪ Otitis media especially in children ▪ Others: Reye’s syndrome, myositis, myocarditis Diagnosis • Specimens: nasopharyngeal swabs, washes, or aspirates • DFA, EIA, Optical Immunoassays, Cell Cultures, Commercially available rapid kits • Culture using amniotic cavity of embryonic chicken eggs, PMK, MDCK • Specific identification of Flu strain: ▪ Immunofluorescence or inhibition of hemadsorption or hemagglutination with specific antibodies Prevention • Vaccination (trivalent vaccine) – best way; administered every year ▪ 2 strains of Influenza A and 1 strain of Influenza B • Annual vaccination for high risk groups • Vaccination (live attenuated or inactivated forms) • Benefits of vaccination: ▪ Reduction of influenza related respiratory illnesses ▪ Reduction of hospitalizations and deaths ▪ Reduction of otitis media

▪

Reduction of work absenteeism

Treatment • Amantadine and rimantadine (administered within 48 hours of infection) ▪ H3N2 - documented resistance to amantadine • Neuraminidase inhibitors (zanamivir, oseltamivir) - more expensive

FAMILY RHABDOVIRIDAE • Rhabdo – “bullet-shaped” • ssRNA: minus strand • Helical • Genus: Lyssavirus ▪ Species: Rabies virus • Causes rabies (literally means “madness”) • Broad range of hosts ▪ Infects all types of mammals • Street virus – isolated from humans and animals • Virus Fixe (Pasteur) – only infects brain tissue • The genus Lyssavirus has 7 genotypes ▪ Human pathogens: genotypes 1-7 ▪ Genotype 1: classic, worldwide type; dogs and foxes ▪ Genotype 2-7: bats • Children are at risk ▪ More common during the summer where there are no classes • Major public health problem; zoonotic disease with many animal reservoirs • Acute infection of the CNS (very fatal) ▪ Once symptoms are present, death is inevitable ▪ Symptoms appear when virus is in the brain ▪ Receptor: ACh receptors • MOT: transmitted by a bite, scratch of a rabid animal (dogs or cats), organ transplant (corneal transplants), exposure of mucosal surfaces to animal’s or patient’s body fluids, inhalation of aerosolized virus; inoculation through intact mucous membrane ▪ Virus is widely distributed in the nervous system, saliva, urine, lymph, milk and blood of infected animals ▪ Scratches – inoculation of virus found on the animal’s claws (due to claw licking) Pathogenesis • G glycoprotein – peplomer; virulence factor ▪ Responsible for the neuroinvasiveness of the virus K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• •

•

Receptor: acetylcholine receptors Initial site of infection – connective tissue and muscle where bite occurred (replication) ▪ Virus moves slowly upward into the brain via the nerve fibers ▪ Proximity, severity, and immune status of the bite to the CNS determine incubation period ▪ Reaches CNS for further replication; encephalitis and neuronal degeneration occur ▪ Dissemination to other organs also occurs Rabies is fatal once clinical disease is seen ▪ Only 2 reported cases with recovery

Clinical Manifestations in Humans STAGE

SYMPTOMS

Prodromal Phase

2-3 days Fever, vomiting, loss of appetite, headache, pain at the site of bite Autonomic nervous system is affected Salivation and sadness are noted

Anger Stage

Clinical Manifestations in Dogs STAGE

SYMPTOMS

Prodrome Stage

Increased alertness or apathy Fever, papillary dilatation Increased muscular tone

Excitement Stage

Paralytic Stage

Death

•

Unusual restlessness Biting at inanimate objects, aimless running Difficulty in swallowing Change in bark or growl Dog unable to take food or swallow water (hydrophobia) Paralysis of the jaw and tongue, drooling saliva Paralysis of hindquarters Within 3-7 days after the initial symptoms

Forms of rabies in dogs: a. Furious rabies – also known as excitatory rabies; more common b. Dumb type – opposite of furious type; lethargy and docility

Neurological Stage or Paralytic Stage

Restlessness and irritability Aggressive Seizures may develop Lasts for 2-4 days Paralysis beginning in the bitten area Difficulty in swallowing; uncontrolled movement, confusion, delirium Hydrophobia, aerophobia Anxiety and hyperactivity Followed by coma and death

• •

•

CPE: eosinophilic cytoplasmic inclusions (Negri bodies) Incubation period: 2-16 weeks or longer ▪ Majority of cases (90-95%) – 1 year or less ▪ Few cases (5-10%) – 1-5 years Length of incubation period depends on: ▪ Severity of the bite ▪ Distance of the bite to the CNS ▪ Viral load ▪ Immune status

Prevention • Pre-exposure prophylaxis – for high-risk individuals (e.g., veterinarians) • Post-exposure prophylaxis by vaccination - 100% effective ▪ Vigorous cleaning of the wound, providing human rabies immunoglobulin, and administering three-injection series of the rabies vaccine • Vaccines available in the Philippines (active immunization): ▪ PVRV: Purified Verocell Rabies Vaccine ▪ PDEV: Purified Duck Embryo Vaccine K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

•

▪ PCECV: Purified Chick Embryo Cell Vaccine Passive immunization: ▪ Rabies Immune Globulin (either HRIG [human rabies immune globulin] or ERIG [equine rabies immune globulin]) ▪ Dose depends on body weight Regimen of vaccination (WHO Standard Intramuscular Regimen) ▪ 5 doses at day 0, 3, 7, 14, 28 ▪ Administered at deltoid area or anterolateral aspect of the thigh of infants ▪ Contraindication: allergy to eggs

Diagnosis • Dogs ▪ ▪

•

Specimen: brain tissue (autopsy) Gold standard – direct fluorescent antibody test (fastest and most sensitive) o Impression smears are made from brain tissue (hippocampus, pons, cerebellum, and medulla oblongata) Seller’s test (direct microscopic examination) Mouse inoculation test RT-PCR

▪ ▪ ▪ Humans ▪ Fluorescent antibody test on brain or skin (taken from the hairline at the back of the neck), or corneal impressions ▪ Culture - suckling/young adult mice, murine neuroblastoma ▪ PCR ▪ Serology (Serum Rapid Fluorescent Focus Inhibition Test) o EIAs - most sensitive ▪ Histology

ARBOVIRUSES • Arthropod-borne viruses ▪ Viral infections that require arthropods (i.e., mosquitoes, ticks, and sandflies) to transmit the disease to susceptible humans ▪ Arthropods – act as vectors ▪ Hosts: man and other vertebrates o Man can either be a dead-end host or a reservoir host; depends on level of viremia o Dead-end host – low level of viremia; hosts that present with the disease o Reservoir host – high level of viremia

•

•

•

Three clinical syndromes of Arboviruses: ▪ Fever with myalgias, arthralgias, and nonhemorrhagic rash ▪ Encephalitis – deadly ▪ Hemorrhagic fever Major Arboviruses: a. Bunyaviridae – Bunyavirus b. Flaviviridae – Flavivirus c. Togaviridae – Alphavirus d. Reoviridae – Orbivirus Medically important Arboviruses: ▪ Yellow fever ▪ Dengue ▪ Chikungunya ▪ Japanese B encephalitis ▪ St. Louis encephalitis ▪ Western equine encephalitis ▪ Eastern equine encephalitis ▪ Russian spring-summer encephalitis ▪ West Nile fever ▪ Sandfly fever

FAMILY BUNYAVIRIDAE • Segmented, minus sense ssRNA, helical, enveloped ▪ Genome has three segments (S, M, and L segments) o S segment – codes for nucleocapsid o M segment – codes for the glycoprotein o L segment – codes for the RNA-dependent RNA polymerase • Majority: vector-borne (replicate in the insect’s gut then appears in saliva) ▪ Some members are rodent-borne (Robovirus) • Disease manifestations: ▪ Febrile illness, hemorrhagic fever or encephalitis ▪ Infection starts as fever, muscle pain, joint pain and photophobia ▪ Mental status changes occur ▪ Rashes, ecchymoses are also seen ▪ Bleeding in the gums, nose • Vector-borne viruses: ▪ Genus Orthobunyavirus ▪ Genus Phlebovirus ▪ Genus Nairovirus • Target organs ▪ Rift Valley Fever – brain and liver (encephalitis and hepatitis) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪

La Crosse, California Encephalitis and Crimean Congo Hemorrhagic Fever – vascular endothelium and liver

Orthobunyavirus • California Encephalitis Virus and La Crosse Virus ▪ La Crosse hemorrhagic fever - summer flu/summer cold • Transmitted by mosquitoes (Aedes) • Causes encephalitis • Common in the United States Phlebovirus • Rift Valley Fever Virus ▪ Disease: Rift Valley Fever – encephalitis and hepatitis ▪ Transmitted by ticks (Ixodes); aerosols of the virus • Sandfly Fever (Phlebotomus fever/Pappataci Fever/3-day fever) ▪ Transmitted by sandflies ▪ Endemic in Europe, North Africa, Asia, and South Africa Nairovirus • Crimean Congo Hemorrhagic Fever virus ▪ Transmitted by ticks (Ixodes) ▪ High mortality rate ▪ Fever, myalgia, arthralgia, and photophobia, mental status changes FAMILY FLAVIVIRIDAE • Plus sense ssRNA, enveloped, icosahedral • Vector-borne; zoonotic viruses ▪ Primarily transmitted by vectors and ticks • Genus: Flavivirus ▪ Japanese Encephalitis Virus - most common cause of arboviral encephalitis in the world ▪ Dengue Virus ▪ West Nile Virus ▪ Yellow Fever Virus ▪ St. Louis Encephalitis Fever • Disease manifestations: ▪ Fever, headache, muscle pain and exanthems, encephalitis, sometimes hemorrhagic fever can occur depending on the virus

Viral Species

Transmitting Vector

Syndrome

Dengue

Mosquito (Aedes, Stegomyia)

Dengue syndrome, DHF, DSS

Yellow fever

Mosquito (Aedes)

Hemorrhagic fever

Japanese B encephalitis

Mosquito (Culex)

Encephalitis

St. Louis encephalitis

Mosquito (Culex)

Encephalitis

West Nile fever

Mosquito (Culex), ticks (Argasidae)

Dengue syndrome, encephalitis

Ticks (Ixodes)

Encephalitis

Tickborne encephalitis (Central European [springsummer meningoencephalitis] and Russian)

Yellow Fever Virus • Causes Yellow Fever • Transmitted by Aedes aegypti or Haemagogus ▪ A. aegypti – vector in urban areas ▪ Haemagogus – vector in jungle/forest • Cases seen in Africa and South America • Manifestations: ▪ Acute infection of fever, myalgia, backache, anorexia, vomiting (black vomit/hematemesis), nausea ▪ Toxic phase: development of jaundice ▪ Distinct life cycles: Jungle Yellow Fever and Urban Yellow Fever a. Jungle/sylvatic yellow fever – monkeys as reservoirs; Haemagogus vector b. Urban yellow fever – humans as reservoirs; Aedes as vector c. Intermediate yellow fever - occurs in small villages in Africa; mosquitoes can transmit yellow fever virus from monkeys to humans, and vice versa • Live attenuated vaccine is available St. Louis Encephalitis • Most common flavivirus infection in the US (Eastern and Central States) • Most have inapparent illness • Initial symptoms: fever, headache, nausea, vomiting and tiredness • Neuroinvasive disease – encephalitis (adults) • Infection is milder in children K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

Transmitted by bird-biting Culex mosquitoes

West Nile Virus • Causes febrile illness, encephalitis, or meningitis • Transmitted by Culex mosquitoes • Documented in Europe, Middle East, Africa, India, Australia, US ▪ Common during the summer months ▪ Reservoir: birds ▪ Humans are dead-end hosts, as the virus does not replicate in humans as well as they do in birds • Risk factor: age (50 years old) •

•

B.

Classic infection: ▪ Fever, headache, fatigue ▪ Skin rashes, swollen lymph glands, and/or eye pain ▪ Primary risk factor for neuroinvasive (meningitis/encephalitis) disease - age greater than 50 years Diagnosis ▪ IgG and IgM ELISA (presence of IgM in CSF indicates CNS infection) ▪ PCR ▪ Antibody neutralization assays for confirmation

Japanese Encephalitis • Leading vaccine-preventable cause of encephalitis in Asia and the Western Pacific region • Vector: Culex tritaeniorhynchus (night biter) • IP: 5-15 days after the mosquito bite • Occurs in rural and agricultural areas (rice farming); more common during the rainy season ▪ Mosquito breeds in rice paddies • S/S: fever, chills, headache, fatigue, nausea, and vomiting; may progress to encephalitis • Vaccine is available Dengue Virus • Five serotypes (DENV1-DENV5) • Most rapid spreading mosquito-borne disease in the world (WHO) • Vector: Aedes aegypti (1o vector) and Aedes albopictus (2o vector; backup) = two most important vectors of dengue; day-biters • Clinical manifestations A. Classic Dengue (Breakbone fever)

Flu-like illness (high fever of 39-40o C after 3-14 days from infection, myalgia, malaise, headache) o Maculopapular rash, leukopenia, lymphadenopathy, thrombocytopenia (less than 150,000/mm3), increasing Hct (5-10%) o Increase in Hct is relative (decreased plasma) o Rarely fatal; few sequelae Dengue Hemorrhagic Fever o More severe and sometimes fatal o Similar to Classic Dengue but with shock and hemorrhage o Secondary infection with another dengue virus serotype o Criteria (WHO): a. Fever or recent history of fever lasting 2-7 days b. Hemorrhagic manifestation c. Thrombocytopenia (< 100,000/mm3) d. Evidence of increased vascular permeability i. Elevated Hct ("≥ 20% above the mean Hct) ii. Hypoproteinemia or hypoalbuminemia iii. Decline in Hct (after volume replacement) Dengue Shock Syndrome o Occurs if the criteria for DHF is present o Evidence of circulatory failure: o

C.

a. b. •

Rapid, weak pulse and narrow pulse pressure ("≤ 20 mmHg [2.7 kPa]) Hypotension for age, restlessness, and cold, clammy skin

Laboratory findings: ▪ In dengue endemic areas: o + tourniquet test o Leukopenia (WBCs ≤ 5000 cells/mm3) o Both criteria helps in making an early diagnosis of dengue infection ▪ Total WBC – usually normal at the onset of fever; then leukopenia develops with decreasing neutrophils and lasts throughout the febrile period ▪ Mild thrombocytopenia (100,000-150,000 cells/mm3) is common ▪ < 100,000 cells/mm3 – seen in ½ of DF patients ▪ Mild rise of Hct (around 10%) o Due to dehydration (high fever, vomiting, anorexia, and poor oral intake) ▪ Elevated liver enzymes (AST)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

•

•

Laboratory methods: ▪ Cell culture – confirmatory o Acute serum sample is needed (0-5 days) ▪ Nucleic acid amplification tests (NATs) o RT-PCR ▪ Serology (IgM/IgG ELISA) ▪ Detection of antigens (NS1 [nonstructural protein1] antigen) o Detectable within 24 hours and up to 9 days following onset of symptoms o Overlaps with viremia; NS1 is detectable prior to IgM seroconversion o For acute DV infection ▪ Rapid methods (ICT methods) Infection due to one serotype confers lifelong protection ▪ Reinfection with a different serotype = more severe form of dengue fever o Especially if DENV1 is followed by DENV2 or 3 o Or if DENV3 is followed by DENV2 ▪ Secondary infections are more severe due to antibody dependent enhancement o Occurs when preexisting antibodies present in the body from a primary (first) dengue virus (DENV) infection bind to an infecting DENV particle during a subsequent infection with a different dengue serotype. The antibodies from the primary infection cannot neutralize the virus. Instead, the Ab–virus complex attaches to receptors called Fcγ receptors (FcγR) on circulating monocytes. The antibodies help the virus infect monocytes more efficiently. The outcome is an increase in the overall replication of the virus and a higher risk of severe dengue. Circulating strains in the Philippines – DENV2 and DENV3

Zika Virus • 1st identified in Uganda and Tanzania in 1952 ▪ From the Zika Forest • MOT: Mosquito bite (Aedes); sexual contact; exposure to infected blood vertical transmission ▪ Same vectors as dengue (A. aegypti and A. albopictus) • S/S: similar to DF; usually self-limiting ▪ Fever, skin rashes, conjunctivitis, muscle and joint pain, malaise, and headache ▪ Mild ▪ Last for 2-7 days

•

•

Possible complications ▪ Microcephaly and mental retardation (many cases in Brazil) ▪ Autoimmune disease ▪ Guillain-Barre syndrome (demyelination) No vaccine

FAMILY REOVIRIDAE • “Reo” – Respiratory Enteric Orphan Virus • Segmented (12 RNA segments), dsRNA • Naked, icosahedral • Causes Colorado Tick Fever (Coltivirus, subfamily Spinareovirinae) ▪ Transmitted by ticks (Dermacentor andersoni) whose hosts include deer, squirrels, and rabbits ▪ Dengue-like infection in the Western United States ▪ S/S: fever, myalgia, nausea, vomiting, rarely encephalitis, photophobia, myalgia, arthralgia, and chills FAMILY TOGAVIRIDAE • Plus-sense ssRNA, enveloped, icosahedral • Genera: a. Alphavirus (Arbovirus): vector-transmitted b. Rubivirus (causes German measles/Rubella/3-day fever) • Peplomer glycoproteins are hemagglutinins Alphavirus • Cause a variety of mosquito-borne diseases that usually cause encephalitis 1.

2. 3.

4.

Eastern Equine Encephalitis ▪ Occurs in the Eastern US ▪ High fatality rate; half of those who survive suffer permanent CNS damage ▪ Vector: Culex, Aedes ▪ Natural hosts: birds ▪ Dead-end hosts: humans and horses o Hosts that manifest the disease Western Equine Encephalitis – milder than EEE Venezuelan Equine Encephalitis ▪ Mortality is less common ▪ Encephalitis seen more in children Chikungunya ▪ Kimakonde language meaning, “to become contorted” (patients are stooped) ▪ Transmitted by Aedes aegypti and Aedes albopictus K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪ ▪

▪ ▪ ▪ ▪

▪

Arthritis and arthralgias S/S: appear between an average of 4 and 7 days (2-12 days) after the patient has been bitten by the infected mosquito: a. High fever (40o C/104o F) b. Joint pain (lower back, ankle, knees, wrists, or phalanges), joint swelling ♦ May last for weeks or even years ♦ Fever and arthritis – predominant symptoms c. Rash, headache, muscle pain, nausea, fatigue Usually affects adults Rarely fatal; self-limiting Infection confers immunity Diagnosis: a. Serology (IgM/IgG): IgM levels are elevated in acute infections b. Culture c. PCR Laboratory findings: o Lymphopenia (low lymphocyte count) o Thrombocytopenia o Elevated creatinine (muscle damage) o Elevated liver enzymes

FAMILY BUNYAVIRIDAE (Rodent-Borne) • Rodent-borne (in contrast to other genera which are arthropod-borne) Hantavirus (species: Hantaan virus, Seoul virus, Puumala virus, and Dobrava virus) • Hemorrhagic fever with renal syndrome • 1950s: Korean war ▪ American soldiers were the first affected •

•

Old World Hantaviruses - endemic to Europe and Asia ▪ Puumala Hantavirus – causes nephropathia epidemica ▪ Reservoir host – deer mouse ▪ MOT: inhalation of aerosols of urine, feces and saliva of infected rodents New World Hantaviruses - in the Americas ▪ Sin Nombre, Black Creek Canal, Bayou, Monongahela, New York, Oran, Andes, Lechiguanas, Laguna Negra

Sin Nombre Virus • Sin nombre – “no name”; formerly the Muerto Canyon virus • Newly identified virus in the American Southwest • Causes Hantavirus Pulmonary Syndrome • MOT: inhalation of aerosolized mouse urine, saliva, or feces • Host - deer mouse (Peromyscus maniculatus) • S/S: fever, chills, myalgia, hypotensive shock may also occur; DIC, hemorrhagic fever, kidney disease, and acute respiratory failure • Diagnosis: EIA, immunohistochemistry Bayou Virus - carried by the rice rat (Oryzomys palustris) Black Creek Canal Virus - carried by the cotton rat (Sigmodom hispidus) New York-1 Virus - carried by the white-footed mouse (Peromyscus leucopus) ELISA - method of choice for diagnosis (detection of virus-specific IgM or IgG antibodies)

FAMILY TOGAVIRIDAE (Non—arthropod-borne) Rubivirus • Rubella virus • Causes Rubella/German measles/3-day measles/tigdas hangin ▪ Highly contagious viral infection of children and young adults; milder than measles ▪ MOT: coughing and sneezing (respiratory droplets); found in saliva ▪ IP: 2-3 weeks • S/S: ▪ Low-grade fever ▪ Maculopapular rashes lasting for 3 days o Begins on the face, then spreads ▪ Mild conjunctivitis, nausea, posterior auricular and sub occipital lymphadenopathy, arthritis (adults and women) ▪ Forscheimer spots (exanthema consisting of petechial lesions on the soft palate) • Congenital Rubella Syndrome ▪ Problem in pregnant women ▪ Infection of pregnant women in the 1st trimester particularly 1st month ▪ May cause congenital malformations on the unborn child o Patent ductus arteriosus, cataracts, deafness and mental retardation, premature birth, microcephaly K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

•

▪ Teratogen (toxic/dangerous to unborn child) ▪ Worse when infection occurs in the earlier stages of pregnancy Prevention: ▪ MMR vaccine (attenuated vaccine) ▪ 2 doses o 1st dose: 12-15 months o 2nd dose: 4 or 6 years old ▪ Persons who are working in hospitals, soon to be pregnant women or women who are in their child-bearing age should be vaccinated (1 or 2 doses) Diagnosis ▪ Culture ▪ Serology (ELISA, hemagglutination inhibition) o Any rubella antibody is protective o Solid-phase and passive hemagglutination tests - most sensitive ▪ PCR

•

• •

▪ Disease lasts for 3-8 days ▪ Complications: dehydration and metabolic acidosis Diagnosis ▪ Direct methods (EM) ▪ Serology (IgM/IgG) ▪ ELISA ▪ PCR Prevention: wash hands; basic sanitation and hygiene Vaccination: given orally in 3 doses beginning at age 6-12 weeks ▪ RotaTeq - human-bovine rotavirus vaccine ▪ Rotarix

FAMILY REOVIRIDAE (Non—arthropod-borne) • dsRNA, naked • Double capsid structure • Respiratory Enteric Orphan Virus • Genera: ▪ Rotavirus – most important cause of viral gastroenteritis among infants o Death due to severe dehydration Rotavirus • Most important cause of infant gastroenteritis in the world (most common cause of viral gastroenteritis in infants and children) • Five groups (A, B, C, D, E) ▪ A – most frequent • Causes death and failure to thrive among infants • Leading cause of death for children under 5 years old • MOT: oral-fecal route; more common in developing countries • IP: 1-3 days • S/S: fever, abdominal pain, vomiting, watery diarrhea, dehydration • Pathology ▪ Affects villi of small intestine ▪ Impairs transport mechanism of nutrients ▪ Diarrhea is due to impaired sodium and glucose absorption K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

HEPATITIS VIRUSES VIRAL HEPATITIS • Systemic disease primarily involving the liver • Most causes of acute viral hepatitis in children and adults are caused by one of the following agents: a. HAV – infectious/epidemic hepatitis b. HBV – serum hepatitis c. HCV – post-transfusion/non-A non-B d. HDV (Delta Virus) e. HEV – enteric non-A non-B hepatitis • MOT a. HAV and HEV – oral-fecal b. HBV, HCV, and HDV – parenteral, needle-stick, sexual contact, IV drug use • Other viruses implicated in cases of sporadic hepatitis: ▪ Yellow Fever virus ▪ CMV ▪ EBV ▪ HSV ▪ Rubella ▪ Enteroviruses • Hepatitis viruses produce acute inflammation of the liver, resulting in clinical illness ▪ Targets the hepatocytes ▪ Bile ducts are not extensively affected ▪ S/S are similar regardless of the etiology Pathogenesis • Early S/S: ▪ Flu-like illness (fever, nausea, vomiting, myalgia, diarrhea) ▪ Mild to moderate pain on RUQ of the abdomen; tender pain (painful when touched) • Progression: ▪ Hepatomegaly, liver tenderness, jaundice, dark-colored urine, light-colored feces ▪ Elevated: bilirubin and liver enzymes (AST and ALT) o Ballooning of liver cells = rupture = release of enzymes ALT and AST

Lining of bile canaliculi is obstructed = release of enzymes ALP and GGT (mild elevations in chronic cases) Regardless of the etiology, identical histopathologic lesions are observed in the liver during acute disease o Ballooning degeneration of cells o Dying hepatocytes contain eosinophilic Councilman bodies o

▪

Clinical Forms of Hepatitis A. Acute Hepatitis – typical form with associated jaundice ▪ 4 phases (incubation, pre-icteric, icteric, convalescence) B. Fulminant – severe hepatitis; liver failure = death C. Subclinical Hepatitis without jaundice ▪ With demonstrable antibody but no history of hepatitis D. Chronic (!≥ 6 months) ▪ Hepatitis B and Hepatitis C ▪ May lead to liver carcinoma ▪ Long-term and persistent ▪ Biliary obstruction – jaundice Properties of Hepatitis Viruses Hepatitis A

Hepatitis B

Hepatitis C

Hepatitis D

Hepatitis E

Family

Picornavirid ae

Hepadnaviridae

Flaviviridae

Unclassifie d

Hepevirida e

Genus

Hepatovirus

Orthohepadnavi rus

Hepaciviru s

Deltavirus

Hepevirus

Virion

Icosahedral

Spherical; Dane particle

Spherical

Spherical

Icosahedral

Envelope

No

Yes (HBsAg)

Yes

Yes (HBsAg)

No

Genome

ssRNA

dsDNA

ssRNA

ssRNA

ssRNA

Genome size (kb)

7.5

3.2

9.4

1.7

7.6

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Stability

Heat- and acid-stable

Acid-sensitive

Acidsensitive

Parenteral

Parenteral

Heat-stable

•

Transmissio n

Fecal-oral

Prevalence

High

High

Moderate

Low, regional

Regional

Fulminant disease

Rare

Rare

Rare

Frequent

In pregnancy

Chronic disease

Never

Often

Often

Often

Never

Incubation Period

28 days (1 month)

10-12 weeks (3 months)

4-12 weeks

No

Yes

Yes

Oncogenic

Parenteral

Ethersensitive, acidsensitive

Fecal-oral

2-9 weeks

?; occasional chronic infections

▪ Food-borne or water-borne outbreaks ▪ IV drug use ▪ International travel (countries with poor sanitation) Incubation period: 28 days (1 month)

No

HEPATITIS A • Family Picornaviridae • ssRNA, icosahedral, enveloped • Formerly classified as Enterovirus 72, now Genus Hepatovirus • 7 genotypes (1-7) o Most strains belong to genotype 1 • Only 1 serotype o Monovalent vaccine • No antigenic cross-reactivity with HBV and other hepatitis viruses • Only humans are affected • MOT: almost always transmitted via the fecal-oral route ▪ Ingestion of contaminated shellfish or other food and water ▪ Outbreaks can occur • Children – most frequently infected • Risk factors ▪ Sexual contact with infected person ▪ Household contact with infected person ▪ Daycare products

Clinical Features • Children (< 5 years) – usually asymptomatic • Adults – manifest the disease (mild to severe prolonged) ▪ Jaundice occurs after the viremia • No chronic infection • Symptoms usually last for 2 months • Virus is shed in the feces • Usually self-limiting and non-fatal • Infection provides lifelong immunity Diagnosis • Serology – detection of anti-HAV ▪ IgM anti-HAV – recent infection (acute hepatitis A); infectious ▪ IgG anti-HAV – past infection; indicates immunity to the infection • Culture is not practical • Serology is the most practical method for diagnosis

! •

Virus is shed in the feces even before the onset of jaundice K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Prevention and Treatment • Administration of immune globulin to exposed individuals ▪ 80% effective if given before or early in the incubation period ▪ Given 2 weeks after exposure ▪ Short term (3 months) •

▪ ▪

Prevention ▪ Hepatitis A vaccine (2 doses) ▪ Indicated for: o Infants aged 12-23 months o High-risk individuals o Individuals travelling to high-risk areas Sanitation Chlorination

HEPATITIS B • Family Hepadnaviridae • DNA virus; enveloped • 8 genotypes ▪ Genotype A – longer survival; more resistant; less severe disease ▪ Genotype B – usually seen in patients that eventually develop liver cancer ▪ Genotype C – severe disease ▪ Genotype D – fulminant hepatitis • Virus can survive and retain its viability in the environment for 7 days ▪ Very resistant • Three forms: a. Spherical (disk) particle – possesses the HBsAg b. Filamentous form – elongated; also possesses the HBsAg c. Dane particle – complete; infectious o Intact HBV virion o Outer envelope + inner icosahedral core (nucleocapsid) o Genome: partially double-stranded HBV DNA + viral DNA polymerase • MOT – blood-borne, sexual, perinatal • High risk groups: ▪ IV drug users ▪ MSMs ▪ Individuals from endemic areas o Philippines – children have worse manifestations of HB infection

•

▪ Sexual contact with infected persons ▪ Healthcare personnel ▪ People with tattoos and piercings ▪ Infants born to HBV(+) mothers Incubation period – 10-12 weeks (3 months)

Clinical Features • Duration of symptoms: up to 6 months • Manifestations are similar to Hepatitis A but are more severe and may be life-threatening • Acute hepatitis – usually asymptomatic ▪ Some may experience flu-like illness ▪ < 1% may develop fulminant hepatitis • Chronic carrier state – virus still present after 6 months (positive for HBsAg) ▪ Persistent infection of hepatocytes ▪ Dependent on the levels of T-cytotoxic cells ▪ High risk for the development of hepatocellular carcinoma (due to chronic inflammation) • High risk for chronic HBV infection: ▪ 80-90% of infected infants will eventually develop chronic Hepatitis B infection; give vaccine immediately after post-partum ▪ 30-50% of children infected before 6 years of age ▪ Adults - < 5% o From the 5%, 20-30% will develop liver cirrhosis or liver cancer • Recovery: majority of adults (95%) recover completely ▪ As age increases, the risk decreases • Areas with the highest prevalence for chronic HBV infection: ▪ Africa ▪ Southeast Asia Diagnosis • Clinical presentation (jaundice) • Serology a. HBsAg – detected in high levels of acute or chronic HBV o Also known as Australia antigen o 1st marker that will rise in infection b. HBeAg – product of nucleocapsid; indicates active viral replication; patient is very infectious o Infected person has high levels of HBV; high likelihood of transmissibility c. Anti-HBe – seroconversion; indicates lower levels of HBeAg; lower infectivity K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

d.

e. f.

Total-anti HBc – lifelong marker; persists throughout life o IgM anti-HBc – recent infection (6 months or less); acute infection Anti-HBs – recovery and immunity; develops in vaccinated persons o Marker of immunity HBcAg – not detectable in serum; forms the nucleocapsid core o Left behind in the liver; detected by liver biopsy

Serologic Marker

Result

Intepretation

HBsAg Anti-HBc Anti-HBs

-

Susceptible (no previous exposure)

HBsAg Anti-HBc Anti-HBs

+ +

Immune due to natural infection

HBsAg Anti-HBc Anti-HBs

+

Immune due to Hepatitis B vaccination

HBsAg Anti-HBc IgM anti-HBc Anti-HBs HBeAg

+ + + +

HBsAg Anti-HBc IgM anti-HBc Anti-HBs

+ + -

Acute infection

Chronically infected

1. HBsAg Anti-HBc Anti-HBs

+ -

2. 3. 4.

Four possibilities: Resolved infection (most common) False-(+), still susceptible Low level chronic infection Resolving acute infection

•

•

Chronic infection (low infectivity) ▪ HBsAg (+) ▪ Anti-HBc (+) ▪ Anti-HBe (+) Chronic infection (high infectivity) ▪ HBsAg (+) ▪ HbeAg (+) ▪ Anti-HBc (+)

Treatment, Prevention, and Control • Treatment ▪ Acute hepatitis: no antiviral therapy is typically used ▪ Chronic hepatitis: inteferons (peginterferons, α-2a or peginterferon α-2b), nucleoside analogues (lamivudine, adefovir) • Prevention ▪ Blood screening ▪ Vaccination (recombinant vaccine) – 3 doses ▪ Hepatitis B immune globulin – post-exposure prophylaxis

HEPATITIS C • Non-A, non-B (NANB) hepatitis agent • ssRNA, +-sense • family Flaviviridae, genus Hepacivirus K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• • • •

6 genotypes: ▪ Genotypes 1a and 1b – most common in the US Usually related to blood transfusions (also dialysis, IV drug use) Most cases of post-transfusion NANB hepatitis were caused by HCV MOT ▪ Injection drug use (common MOT in US) ▪ Transfusion of infected blood ▪ Organ transplants ▪ Needlesticks ▪ Birth to an HCV (+) mother ▪ Sexual contact (not efficient)

Clinical Features • Symptoms appear 4-6 weeks after infection • Most patients are asymptomatic • Acute form – similar to Hepatitis A and B but inflammatory response is less and symptoms are milder • Chronic form – more prevalent than HBV, often leads to cirrhosis ▪ Most patients (70-90%) develop chronic hepatitis ▪ 20-30% of which develop cirrhosis, which could further lead to hepatocellular carcinoma • Infects liver cells primarily • Mediated by T-cytotoxic cells • Viral replication is enhanced by a liver-specific micro-RNA (enhances production of viral mRNA) • Alcoholism – enhances rate of liver carcinoma Diagnosis • Serology ▪ Anti-HCV is not protective ▪ HCV infection does not confer persistent and lifelong levels of the antibody ▪ Anti-HCV appears 6 weeks or 12 weeks after infection • Chemistry: ALT elevations • Methods: ▪ Gene amplification to detect HCV RNA ▪ EIA (ELISA) – screening test ▪ 2nd generation immunoblot tests – RIBA (Recombinant Immunblot Assay) – confirmatory

!

Treatment, Prevention, and Control • IFN-α and ribavirin – the only effective treatment (50% response rate) • Liver transplant • Supportive • Blood screening • No vaccine available

HEPATITIS D • Delta agent: HDAg surrounded by HBsAg envelope ▪ In blood, HDV (delta agent) contains delta antigen (HDAg) surrounded by an HBsAg envelope • HDV: ssRNA; smallest of known human pathogens • Responsible for about 40% of fulminant hepatitis • Highly dependent on HBV for replication ▪ Defective virus – acquires an HBsAg coat for transmission; will not replicate in the absence of HBsAg • It is often associated with the most severe fotms of hepatitis in HBsAg (+) patients • MOT ▪ Parenteral or percutaneous or mucosal contact with infected blood ▪ Co-infection can occur alongside HBV (Hepatitis B + Hepatitis D) K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Superinfection may also occur (Hepatitis B followed by Hepatitis D) High risk: ▪ IV drug users and hemophiliacs ▪ Agent increases severity of HBV infection ▪ Delta agent carriers – more likely to develop fulminant hepatitis ▪ Vaccination for Hepatitis B confers immunity against Hepatitis D ▪

•

Anti-HBc IgM

HBsAg

Anti-HDV

Anti-HDV IgM

Co-infection

+

+

+

+

Superinfection

-

+

+

N/A

HEPATITIS E • MOT: similar to Hepatitis A ▪ Transmitted enterically and can cause epidemics in developing countries, where water or food supplies are sometimes fecallycontaminated • Target – pregnant women ▪ High chance of mortality during the 3rd trimester Clinical Features • Acute, self-limiting disease similar to HAV • IP: 2-9 weeks • Mortality rate higher than that of HAV • Pregnant women may have a high (20%) mortality rate if fulminant hepatitis develops Diagnosis • ELISA (IgM anti-HEV, IgG anti-HEV)

OTHER HEPATITIS VIRUSES Hepatitis F • Virus-like particles (dsDNA) • Thought to be a mutant strain of HBV

Laboratory Diagnosis • ELISA and RIA (Antigen/Antibody detection) • HDV-RNA detection by PCR • Markers: ▪ HDV – the agent ▪ HDAg – delta antigen (detectable in early acute infection) ▪ Anti-HDV – indicates past (IgG) or present (IgM) infection • Treatment ▪ No known specific treatment ▪ HBV vaccination is protective

Hepatitis G • Flaviviridae • Now called as GBV-C virus • Transfusion-transmitted hepatitis • Related to HCV • No associated disease yet Others • SEN virus – transfusion-transmitted virus

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

▪

HIV

▪ ▪ FAMILY RETROVIRIDAE • RNA viruses that replicate in the nucleus • Requires reverse transcriptase (reverse polymerase/RNA-dependent DNA polymerase) • Some members possess oncogenes that promote carcinogenesis • Some members, such as HIV, are cytocidal (cytotoxic) • Subfamilies: a. Oncovirinae - oncogenic viruses; associated with leukemias, sarcomas, and lymphomas; tumor-causing o HTLV-1 - Tropical Spastic Paraperesis o H T LV- 2 - H a i r y C e l l L e u k e m i a / L e u k e m i c reticuloendotheliosis o HTLV-5 b. Lentivirinae (genus Lentivirus) o HIV-1 o HIV-2 Subfamily Oncovirinae • Human T-cell Lymphotropic Virus (HTLV) • HTLV-1 - first human retrovirus ▪ Causes adult T-cell leukemia, lymphoma, and tropical spastic paraperesis • HTLV-2 - isolated from a patient with hairy cell leukemia Subfamily Lentivirinae • Genus Lentivirinae, meaning “slow” (long incubation; slowly replicating) • Slow viral diseases • HIV-1 - worldwide distribution ▪ Causes AIDS ▪ Common in MSMs (homosexual contact) ▪ Originated from thechimpanzee • HIV-2 - seen in West Africa; less common ▪ Less severe compared to HIV-1 ▪ MOT: heterosexual contact ▪ Originated from the sooty mangabey HIV-1 • Causative agent of AIDS • Genetically related groups

HIV-2 • • • •

Group M - causes the majority of global disease o Divided into 8 clades (subgroups): A, B, C, D, F, G, H, J o Subgroup B - Europe, US, Australia; associated with unprotected oral sex Group O - West Africa Group N - Cameroon

West Africa Less severe 7 subtypes (A-G) MOT - heterosexual contact

Viral Characteristics Structural Components • Capsid ▪ Icosahedral ▪ 2 identical ssRNA pieces ▪ 3 essential retroviral enzymes: 1. Protease - breaks down/cleaves the gag and pol proteins 2. Integrase - enzyme that inserts the viral genome into the host genome 3. Reverse transcriptase • Capsid proteins ▪ p24 - major capsid protein; early marker of HIV-1 infection • Matrix protein (p17) - surrounds the capsid of HIV-1 • Surface glycoproteins (gp) - gp160 (gp120 and gp41) ▪ Located in the envelope ▪ 72 spikes ▪ The entire peplomer is gp160, cleaved inro smaller proteins gp41 and gp120 o gp41 - transmembrane glycoprotein o gp120 - docking protein; interacts with CD4 in Th cells Structural Genes • gag - group specific antigen ▪ Codes for p24 and matrix proteins • pol - polymerase proteins ▪ Codes for reverse transcriptase, endonuclease, and integrase • env - envelope glycoproteins ▪ Codes for glycoproteins that adhere to host cells and produce cytopathic effects (fusion)

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

•

•

▪ p27 vpu gene - viral protein U ▪ Viral assembly and budding ▪ p16 vpr gene - viral protein R ▪ Integration of HIV genome into viral genome ▪ p15 vif gene - viral infectivity factor ▪ p23

PATHOGENESIS OF HIV INFECTION • HIV causes AIDS (acquired immunodeficiency syndrome) ▪ Other names for HIV: AIDS-related virus, HTLV-3 • It was once called the “gay disease” • First recognized among homosexual males who contracted opportunistic infections in the US in 1981 • Discovered in 1981 by Dr. Robert Gallo and Dr. Luc Montagnier

Genes

gag pol

env

Gene Products

Gene Product Function

HIV-1 p55

HIV-2 p53-55

p24 p17 p66, p51

p26 P16 p68

p32 gp160 gp120 gp41

p33 gp125 gp105 gp36

Precursor protein for p24 and p17 Capsid protein Matrix protein Reverse transcriptase component Endonuclease component Precursor protein Docking protein Transmembrane protein

Regulatory Genes • tat gene - transactive transcription gene ▪ Enhances the transcription and expression of viral proteins ▪ p14 • rev gene - posttranscriptional activator ▪ Production of structural proteins ▪ Transports viral mRNA to the cytoplasm of the host cell ▪ p19 Other Genes • nef gene - negative effector ▪ Enhances HIV replication

Routes of Transmission 1. Inoculation by contact with blood ▪ Transfusion of blood and blood products ▪ Needle sharing among IV drug users ▪ Needle-stick injuries, open wound and mucous membrane exposure in healthcare workers (less common than HBV) ▪ Tattoo needles 2. Sexual - most common route ▪ Anal and vaginal intercourse, fellatio ▪ Anal intercourse bears more risk in contracting the disease 3. Perinatal ▪ From HIV-infected mother to fetus ▪ Intrauterine, postpartum, breast milk • • • • • •

Transmitted through body fluids Virus persists indefinitely in infected hosts Viruses have high mutation rates (oncogenicity) Infection progresses slowly through stages It takes years for the disease to develop Host factors such as age, stress, genetics, and concurrent infections are important in the pathogenesis of HIV

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Disinfection and Inactivation • 10% hypochlorite • 50% ethanol, 35% isopropyl alcohol • 0.5% Lysol®, 0.5% paraformaldehyde • 0.3% H2O2 • pH 1.0, pH 13.0 ▪ Readily inactivated in liquids ▪ Dried, proteinaceous materials need increased temperature and increased heating time for inactivation HIV Receptors • CD4 molecule - main receptor for the gp120 in the HIV envelope ▪ Found in T-helper cells and other CD4+ cells such as monocytes/macrophages, dendritic cells, and Langerhans cells • Co-receptors - chemokine receptors; binding to CD4 is not sufficient for infection (co-receptors must also be bound by gp120) ▪ CXCR4 ▪ CCR5 • HIV strains based on the use of chemokine co-receptors a. R5 strains - use CCR5; M-tropic (infects the monocyte/ macrophage lineage) b. X4 strains - use CXCR4 preferentially infects T-helper cells (T-tropic) • In approximately 90% of cases, the R5 (M-tropic) type of HIV is the dominant virus found in the blood of acutely infected individuals and early in the course of infection. Over the course of infection, however, T-tropic viruses gradually accumulate; these are especially virulent because T-tropic viruses are capable of infecting many T cells and even thymic T-cell precursors and cause greater T-cell depletion and impairment Disease Mechanisms • HIV infects CD4+ T cells and cells of the monocyte/macrophage lineage ▪ Monocytes, macrophages, alveolar macrophages, dendritic cells of the skin, and microglial cells • Causes lytic and subsequent latent infection of CD4 T cells and persistent low-level productive infection of macrophage-lineage cells

Disease Manifestations of HIV/AIDS • Three stages: 1. Acute Infection/Acute Retroviral Syndrome/Acute HIV Infection 2. Clinical latency 3. AIDS Acute Infection • 2-4 weeks after exposure (may be 6-8 weeks) • Flu-like illness; some are asymptomatic ▪ Fever, night sweats, sore throat, muscle and joint pain, rash, fatigue • Large amounts of HIV in the blood • Coincides with the replication of HIV in CD4 cells • Patient is very infectious at this stage

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

•

Window period - 6 months

Clinical Latency • Asymptomatic or chronic HIV infection • No symptoms/illness • Low viral replication • Lasts for a decade or less for untreated patients (10-20 years, depending on immune status) • In the middle and end of the phase, there is an increased viral replication and decreased levels of CD4 cells Acquired Immunodeficiency Syndrome (AIDS) • Terminal phase; people living with HIV ▪ Not all HIV-+ individuals develop AIDS; prevent progression by staying healthy and taking anti-retrovirals • Immunocompromised state of the infected person • Patients are vulnerable in contracting opportunistic infections and certain cancers • Definition - CD4 count less than 200/µL ▪ Normal CD4 count - 500-1600/µL • AIDS is also considered if patient suffers at least one opportunistic infection regardless of the CD4 count • Death occurs in 3 years if untreated Demographics • Worldwide: as of 2015, 36.7 million people are living with HIV around the world • Sub-saharan Africa - most affected region in the world • Most common modes of transmission in the Philippines 1. Males having sex with males only (57%) 2. Males having sex with males and females (26%) 3. Male-female sex (14%) 4. Injectional drug use (2%) 5. Mother to child (< 1%) 6. No data (1%) • In the Philippines, most commin in NCR, Region 4A, and Region 7 ▪ In 2018, there are 32 newly diagnosed cases of HIV per day (22 in 2015, 13 in 2013, 7 in 2011, and 2 in 2008) • 2010 and onwards - most predominant cause of HIV in the Philippines is male-to-male sexual contact ▪ 1984-2009 - predominant MOT was male-female sex

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

Pediatric AIDS • Usually presents with clinical symptoms by age 2, followed by death within 2 years • Children are particularly susceptible since the immune system has not developed at the time of primary infection AIDS-Defining Infections (Opportunistic Infections) A. Protozoal ▪ Toxoplasmosis ▪ Cryptosporidiosis ▪ Isosporiasis B. Fungal ▪ Candidiasis ▪ Pneumocystis pneumonia ▪ Cryptococcosis ▪ Histoplasmosis ▪ Coccidioidomycosis C. Viral ▪ CMV, EBV infection ▪ HSV infection ▪ Hepatitis B and C ▪ Varicella-Zoster ▪ Adenovirus infection ▪ Polyomavirus ▪ JC virus D. Bacterial ▪ Tuberculosis ▪ Listeriosis ▪ Nocardiosis ▪ Salmonellosis ▪ Streptococcal infection ▪ Infection by Mycobacterium avium-intracellulare complex Opportunistic Neoplasias • Kaposi’s sarcoma • Primary lymphoma of the brain • Other non-Hodgkin’s lymphomas Others • HIV wasting syndrome • HIV encephalopathy • Lymphoid interstitial pneumonia

LABORATORY DIAGNOSIS Screening Tests • ELISA (detection of only anti-HIV antibodies) a. 1st-generation tests - purified viral lysates or antigens (crude antigens) b. 2nd-generation tests - recombinant viral proteins c. 3rd-generation tests - double antigen sandwich assay d. 4th-generation tests - detection of antigen (p24) and antibody (anti HIV 1 or anti-HIV 2) e. 5th-generation tests - antigen and antibody + differentiation

Generation

1 and 2

3

4

• • •

•

Mechanism Viral lysate used to bind patient HIV antibody. Detects IgG antibody to HIV viral proteins. Second generation assays are the same as first generation, but use purified antigens/ recombinant virus Same mechanism as first and second generations, but IgM is also detected, which decreases the window period Same mechanism as third generation, but in addition uses an antibody to detect p24 antigen in the patient’s serum

Window Period (time between HIV exposure and the point in which the test will give and accurate result)

Example

4-12 weeks

Vironostics HIV-1 Microelisa (bioMerieux, Inc.) Genetic Systems rIAV EIA HIV-1

3-4 weeks

2 weeks

HIV Ab HIV1/HIV2 rDNA Genetic Systems HIV1/HIV2 + EIA

Vidas HIV Duo Ultra

Rapid diagnosis (using serum, plasma, blood, saliva) Other tests: immunochromatography, rapid tests, particle agglutination For ELISA: ▪ If non-reactive: report as negative ▪ If reactive: the test should be done in duplicate o Confirmatory testing using Western blot o For confirmation, send to STD/AIDS Central Cooperative Laboratory (for patient samples) or RITM (for blood products) Must be confidential and with consent

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

• •

HIV-proficient medical technologists - only ones allowed to perform diagnostic tests for HIV Reference laboratories for confirmatory testing: ▪ RITM (Research Institute for Tropical Medicine) - for reactive patient blood products ▪ SACCL in San Lazaro Hospital - for reactive patient samples

Confirmatory Testing • In SACCL and RITM, serum samples are screened twice ▪ Rapid testing (Serodia HIV 1/2) ▪ 4th generation EIA (HIV 1/2 and p24) If positive to either, confirmatory testing using Western blot is performed Western Blot - confirmatory test for HIV infection ▪ Detects antibodies specific to p24, p31, gp41, and gp120/gp160 o gp160 - viral envelope precursor (env) o gp120 - viral envelope protein that binds CD4 o p31 - reverse transcriptase (pol) o p24 - viral core protein (gag) ▪ Positive - 2 out of 3 major antigens detected (p24, gp41, and gp120/gp160 ▪ Negative ▪ Indeterminate - re-test after 6 months ▪ Viral proteins are separated first and immobilized. In subsequent steps, the binding of serum antibodies to specific HIV proteins is visualized ▪

•

There are different criteria for the interpretation of HIV Western blot results (e.g., CDC, WHO, American Red Cross) ▪ The most important antibodies are those against the envelope glycoproteins gp120, gp160, and gp41 ▪ p24 antibody is usually present but may be absent in the later stages of HIV infection Other confirmatory tests - IFA, RIPA, line immunoassays Post-counseling is done once a positive result is released ▪

• •

Plasma Viral Load • Prognostic value • Predictor of long-term clinical outcomes CD4 Lymphocyte Count - best predictor of short-term risk of developing opportunistic disease • AIDS - CD4 is less than 200/µL • Normal ratio of CD4 to CD8 - 2:1 • Ratio of CD4 to CD8 cells in AIDS - 0.5:1 (1:2) • Makes use of immunophenotyping ▪ Principle - flow cytometry ▪ Incubation of whole blood with fluorescent labeled antibodies; lysis of RBCs and stabilization of WBCs with paraformaldehyde ▪ Measures amount of CD4 left in a host ▪ Specimen of choice - EDTA-anticoagulated whole blood Molecular Methods • Nucleic acid testing Testing of Infants • Qualitative HIV1 DNA PCR Test - preferred • Quantitative HIV RNA assays

TREATMENT • Antiretroviral therapy (HIV regimen) • PEP - Post-Exposure Prophylaxis (72 hours) • PrEP - Pre-Exposure Prophylaxis; for high-risk individuals; those who do not use protection when having coitus • There have been cases of drug-resistant HIV strains

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY

PRIONS PRIONS • Proteinaceous infectious particles • Cause transmissible spongiform encephalopathies • Cause neurodegenerative diseases affecting humans and animals • Unique infectious agent (not actually an organism; just a protein- PrPSC) • No genomes (only an abnormal mass of protein capable of altering conformational arrangement of human proteins in vivo) Characteristics • Long incubation time (takes years to manifest) • Gradual increase in severity • No immune response mounted by the host • Non-inflammatory process • Presence of spongiform changes (vacuoles and plaques in brain tissue) • Signs and symptoms - cerebellar ataxia, loss of motor and speech function, memory loss, behavioral problems • Among humans ▪ Creutzfeldt-Jakob disease - familial disease; rapid onset of dementia ▪ Kuru - in Papua New Guinea; contracted by the cannibalistic practice of eating human brains ▪ Gertsmann-Straussler-Scheunker disease - variant of CJD (vCJD) ▪ Fatal Familial Insomnia • Among animals ▪ Scrapie - in sheep; infection is acquired by animals eating feeds mixed with carcasses ▪ Bovine spongiform encephalopathy (Mad cow disease) - mental changes brought about by eating beef • Highly resistant to inactivation ▪ Not inactivated by autoclaving

Diagnosis • Clinical findings • Histopathology • PrPSC immunostaining • Serology • Nucleic acid testing Disease

Typical Syndromes

Etiology

Kuru

Ataxia, myoclonus followed by dementia Dementia, myoclonus followed by ataxia Psychiatric changes, ataxia, dementia, Sleep disturbances followed by dementia Ataxia followed by dementia

Infectious

Sporadic CJD and Familial CJD Variant CJD FFI GSS

Duration before Death Months

Unknown (sporadic) Inherited (familial) Infectious

1 to few years

Inherited

1 year

Inherited

Few years

16 months

Mode of Transmission • Ingestion • Transmission via intake of hormones from pituitary tissue • Dura mater and corneal transplants • Contaminated EEG electrodes

K. MOLBOG, 2019 UNIVERSITY OF SANTO TOMAS MEDICAL TECHNOLOGY