Micro II - fungi, virus, mycobacteria, parasites

Mycobacteria

A family of actinobacteria, genus of acid fast gram positive bacilli - obligate aerobes

TB, leprae, nontuberculous mycobacteria

3 main groups of mycobacteria

Sputum (1st morning is preferred)

Specimens for Mycobacteria - process under the hood (aerosol risk)

Ziehl-Neelsen (Most common), Kinyoun, Flurochrome

Staining methods for Acid-Fast bacilli

Carbol fuschin (primary stain) → alcohol → methylene blue (counterstain) → heat slides in between

Describe the Ziel-Neelsen stain

Carbol fuschin (primary stain) → phenol → methylene blue/Malachite green (counterstain) → COLD FIXED

Describe the Kinyoun stain

AFB yellow-orange fluorescence under UV (faster and more sensitive)

Describe the Fluorochrome stain (Auramine-Rhodamine)

Nucleic Acid Amplification test (NAAT)

Which other test for mycobacterium detects a pathogen’s genetic material by amplifying specific sequences, making them easier to identify (rapid and accurate)?

Interferon-gamma release assays (IGRA)

Which other test for mycobacterium is a blood test that detects tuberculosis (TB) by measure interferon gamma released from T-cells (great for BCG vax peeps)?

get multiple specimens, resistance testing

Considerations for mycobacterium testing

Yeasts (like candida), Molds (aspergilus)

Species of fungi

Fungi

A eukaryotic cell with a chitin-containing wall (similar to mammalian - so watch yo self)

immunocompromised peeps (small population = specific testing sites)

Who are we worried about for opportunistic fungi infections?

superficial, cutaneous, subcutaneous, systemic

Fungal diseases can be…

unicellular, budding, creamy, smooth colonies

Features of Yeasts

Multicellular (hyphae), spore formation (aerosol risk), fuzzy, woolly colonies

Features of Molds

Any tissue or body fluid (hair, skin scrapings, nails, urine, blood tissue, bone marrow)

Specimens for Mycology

ensure proper collection from the proper site, transport quick, process in the hood

Processing of mycology specimens

Wet mount, KOH prep, Calcofluor white + KOH

Direct microscopic exams

Wet mount

A sample placed on a slide, mixed with a drop of saline/sterile water covered with a coverslip that allows for the observation of LIVING cells

KOH (potassium hydroxide)

What can be added to a wet mount to clear the cellular material, but leaves the fungal elements

Stain blue/black/purple, show budding or hyphae forms

What do fungi look like on a gram stain?

India ink

A negative staining method that is used to rapidly identify cryptococcus (halo)

Culture and sensitivities (do one with and without antibacterial/antifungal)

gold standard for diagnosis of mycology based disorders

SAB (sabouraud agar)

Most commonly used media for fungal growth (can be enriched with dextrose or Brain heart infusion)

potato dextrose

Selective media for saprophytic and dimorphic

Czapek dox

Selective media for aspergillus

Inhibitory mold (IMA)

Selective media for dimorphic

niger seed agar

Selective media for C. neoformans

incubate a 30 degrees celsius for 4-6 weeks

Since fungi are obligate - what environment are we growing them at

Assimilation test

What test combines enzyme and activity and ability to use carbon/nitrogen for growth and sometimes fungal resistance

Germ tube

Differentiates candida albicans by the formation of ___________ from yeast cells when incubated in serum

Chromogenic agar

A special growth media that changes colors based on the presence of enzymatic reactions

Catalase (hydrogen peroxide), Oxidase, Urease

Examples of enzyme activity testing for mycology

Nitrate to nitrite reduction, Methyl red test

Tests for the production of acid-end products

parasites

Lives in/on a host at that host’s expense (may have a temporary independent state before transmission)

protozoa, helminths, arthropods

Types of parasites

GI tract, blood, urogenital tract, tissue

Common sites of parasite infections - more often in tropical areas

travelers, immunosuppressed, refugees

Who is at an increased risk of parasitic infections

Stool 3 samples, 1-2 days apart within 10 days (PRIMARY), Blood thin/thick smears (malaria), Tape collection (pinworms), Others based on suspected parasites

Samples for parasites

keep refrigerated, avoid contaminating with water or urine, transport to lab within 24-48 hr

Rules for stool samples in parasitology

Do test right after waking up, get several samples, check under fingernails

Rules for tape collection for enterobius vermicularis

macroscopic (what does the stool look like), wet mounts, concentration technique (spin with zinc sulfate), Staining (reaffirms diagnosis and reveals nuclear material), antigen detection, molecular testing, cellophane tape, blood and tissue smears

Detection methods for parasites

Iodine, trichrome (most common), iron hematoxylin, acid fast

Types of staining for parasites

trophozoites, ova, larvae

What parasites are wet mounts good for

giardia, cryptosporidum

What parasites is antigen detection good for

Malaria (use the giemsa stain), babesia, trypanosoma, loa loa

Blood and tissue smears are good for which parasites - thick has high sensitivity?

Virus

The smallest, obligate infectious agent that requires a host cell to live and reproduces using host machinery for propagation, transmission, and infection - found everywhere (ubiquitous)

Inner nucleic acid core, protein capsid, lipid envelope

Structure of a virus - general

swabs, fluids, tissue samples, nasopharyngeal swab, lavage, stool, urine, CSF, blood, bone marrow, other body fluids

Specimen for viral specimen - collect early and transport in the correct media (specific swabs)

Cell culture (🏆), shell vial assay, antigen detection (rapid testing), molecular testing (PCR), POCT (rapid)

Viral detection methods

incubate for 1-4 weeks with CPE observation

Rules for viral cell cultures

a faster culture with IF

What is a shell vial assay?