MPL 202 Midterm Review 1 PDF - Microbiology

MPL 202 Midterm Review 1 ======================== **Learning Objectives** 1\. Outline the common causes of laboratory errors and how to prevent them.\ 2. Explain how pathogens cause infections, how they progress, and their impact on the host.\ 3. Describe the characteristics of bacteria, viruses,...

MPL 202 Midterm Review 1 ======================== **Learning Objectives** 1\. Outline the common causes of laboratory errors and how to prevent them.\ 2. Explain how pathogens cause infections, how they progress, and their impact on the host.\ 3. Describe the characteristics of bacteria, viruses, fungi, and parasites and how they are detected in patient samples. **Path of lab workflow** - Within the control of primary healthcare providers - Specimen collection during pre-analytical phase - Patient assessment and infection control precautions during post-analytical - Within the control of the lab - During analytical phase - Specimen processing \> extraction \> reagent preparation \> reaction \> results **Errors in Total Testing Process** - Pre-analytical -- **most common for errors to occur** - High workload leads to error - Inappropriate test request - Order entry error - Patient/specimen misidentification - Sample collection ---\> inappropriate site, hemolyzed/clotted sample, insufficient volume (many, may others) - Inappropriate container, handling, storage, and transportation - If not stored properly, sample gets contaminated -- leads to poor/skewed results -- leads to pts being treated for something that's not actually there - **"garbage in = garbage out"** - **\>90% of diagnostic errors occur in the pre- and post-analytical phases of testing** - Analytical (7-13%) - Undetected failure in laboratory QC - Equipment malfunction - Post-analytical - Failure in reporting/addressing report - Excessive turn-around time - Transcription errors - Failure/delay to report critical values - Incorrect physician interpretation **Microbes are ubiquitous within the environment** - Ceiling tiles - Aspergillus and other fungi - Alcohol-based hand rub (ABHR) stations - ABHR kills most but not all pathogens (e.g. Clostridium difficile spores) - High-contact surfaces (fomites) -- MRSA, influenza, other viruses and bacteria - Sinks - Water-borne pathogens (Serratia, Pseudomonas, others...), antibiotic resistant organisms (ARO), biofilms - Understanding Infection Control Practice, pathogen transmission, healthcare-associated infections, epidemiology of disease and diagnostic approaches is necessary to reduce spread of bacteria and disease **Phylogenetic Tree of Life\ **- three domains of life -- Bacteria, Archaea, Eucarya\ - Archaea (extremophiles) have **never** been associated with human infection\ - Eucarya can cause infection in humans -- bugs included Overview of Microorganisms and Other Infectious Agents (Pathogens) A microorganism that can cause disease Viruses Prions Eukaryotes (nucleus) Parasites (e.g. helminthes) Prokaryotes (no nucleus) CELLULAR (LIVING) plasmodia) Malaria Fungi (e. g, tinea) Athlete\'s ftx»t (i\_e\_ bacteria) ACELLULAR (NON.LIVING) Virus (eg HIV) CJD - **Pathogens: a microorganism that can cause disease** - Eukaryotes: often unicellular but can be multicellular - Prokaryotes: unicellular - Prions: Associated with mad cow disease; misfolded proteins hat self-replicate, not exactly viruses - Archaea: generally extremophiles, live in environments with extreme temps, pHs, and salinities - Bacteria, viruses, viroids, and prions can all cause infection. Eukaryotic cells can be microscopic, but they are larger than prokaryotic cells, like bacteria. Prokaryotes are larger than viruses, viroids, and prions **Basic steps for infection to occur** 1. Must have a pathogen 2. Must have a susceptible host 3. Pathogen must gain entry into host (through cuts, mucous membranes, ingestion, breathing etc.) 4. Pathogen must establish colonization in host a. Adhere to host b. Replicate within host c. Evade host defences 5. Pathogen must cause damage to the host d. Direct via virulence factors (e.g. toxins) e. Indirect via host's own immune response **Portals of Entry** - - **Mucous membranes** - Respiratory tract - Gastrointestinal tract - Genitourinary tract - Placenta - **Skin** - **Parenteral** - Bite - Puncture - Injection - Wound - - Most pathogens have preferred portals of entry - Ex: *Streptoccocus pneumoniae* will cause pneumonia when inhaled; when ingested will not - Some pathogens will cause illness no matter how they enter. Ex: Ebola = super infectious; sufficient enough viral load anywhere will cause infection - Some pathogens will cause different disease depending on portal of entry - Ex: *Yersinia pestis* (plague) has 2 forms; bubonic (following flea bite) and pneumonic (following inhalation of aerosolized organisms) - **Exposure:** contact with a potentially infectious agent - **Colonization:** persistence of *microbe* on or within another organism -- not infection, lots of microbes are on us at all times, sometimes they are helpful/don't do harm - **Infection:** persistence of pathogen on or within another organism, with a resulting deleterious effect on the host - **Period of communicability:** period of time that an individual is capable of transmitting infection, can happen before symptom onset - **Latency period:** time from exposure to when individual becomes infectious -- body is fighting disease to see who comes out on top - **Incubation period:** time from exposure to development of symptoms **Human barriers to infection:** - **Microbiota:** ecological community of commensal, symbiotic, and pathogenic microorganisms - Physically blocks access to mucosa below - Repetitive exclusion - **Cilia:** constantly forcing things away (from your lungs, body, etc) - **Mucous:** another physical barrier - **Lysozyme** destroys bacterial cell wall **Pathogen Toolkit: Virulence Factors** - Virulence factors are molecules produced by pathogens enabling them to achieve the following: 1. Colonization of the host a. Adherence: e.g. adhesions (bacteria), attachment proteins (viruses) i. Adherence in bacteria is much larger than attachment proteins in virus b. Destructive enzymes e.g. hylauronidase (destroy connective tissues), hemolysis (destroy cells, including RBCs, immune cells) c. Toxins: e.g. anthrax toxin (bacteria) 2. Immunoevasion, evasion of host immune system d. Prevent antibody binding: e.g. capsule (primarily bacteria, some yeasts) ii. Capsules around the bacteria body act as a cloaking shield to hide from antibodies e. Prevent phagocytosis: e.g. biofilm (bacteria, yeasts) iii. Biofilms are large films that prevent WBC from ingesting them 3. Immunosuppression,inhibition of host immune response f. Destructive enzymes: e.g. immunoglobulin proteases (destroy antibodies) g. Conversion enzymes: e.g. catalase (destroys hydrogen peroxide) iv. Body produces H2O2 to fight infection -- catalase destroys this 4. Invasion of host cells (if pathogen is intracellular) h. Destruction (lysis) of host cells or suppression of their normal activity 5. Obtain/sequester nutrients from host **\ ** **Host damage and infection outcome** - Damage can be direct (e.g. toxin-mediated) or indirect via host immune response (e.g. septic shock) - **Endotoxin:** part of cell wall of some bacteria (lipopolysaccharide; LPS) and released when cells lyse and causes immune response - Triggers immune cells to release cytokines in toxic concentrations - Antibiotics can trigger release, pt may 'get worse before they get better' - **Exotoxin:** enzymes that are secreted by bacteria that perturb some aspect of normal host physiology - Generally very specific to genus/species - Highly immunogenicity: e.g. tetanus toxoid used in vaccines ---\> neutralize toxin ---\> produce antibodies ---\> relay to fight the toxin - Damage to host facilitates invasion, dissemination, and transmission - Ex. *Vibrio cholera* uses cholera toxin to cause diarrhea (spreads out by droplets) **Fundamentals of bacteriology** **Bacteria cell structure** Bacteria Cell Structure Pilus (fimbria) Ribosomes Inclusion Flagellum PlaMnid John Wiley and Sons, Inc.) Capsule or slime layer Cell wall Cell membrane - A cell membrane, surrounded by a cell wall, maybe an extra capsule/ slime layer - Cytoplasm with ribosomes to make protein, a nuclear region where the and floats, and maybe vesicles - External features like flagella and pili - Flagellum propels bacteria cell structure - Plasmids - can carry genes that give bacteria genetic advantages, such as antibiotic resistance. **Bacteria cell wall -- gram +/-** **Gram negative** - 10-20% of cell wall is peptidoglycan - Outer membrane -- 2 membranes and thin cell wall to counter stain - Periplasmic space - Lipopolysaccharide (LPS) **endotoxin** - **Does not absorb dye, so it is pink** **Gram positive** - 60-90% of cell wall is peptidoglycan - **Stains purple, absorbs crystal violet dye** - No outer membrane -- thick cell wall - No LPS **Note:** - Knowing positive vs. negative may be helpful for dicating treatment - The semi-rigid cell wall is outside the cell membrane in bacteria, services 2 functions: - Maintains the characteristic shape of the cell - Prevents the cell from bursting when fluids flow in by osmosis - It is porous, so doesn't help control the flow of nutrients in and out of the cell - It's mostly made up of peptidoglycan, it forms a supportive net around the bacteria, like a chain-link fence - Some types of bacteria may also have teichoic acids, their function remains unclear but are used by bacteriophages (viruses that infect bacteria) to attach and enter the cell **Size and shape of bacteria** - **Prokaryotes:** smallest living organisms - Size: 0.5 to 2 micrometers in diameter - Shape/arrangement - Three shapes: spherical, rod-like, spiral - Coccus -- typical spherical bacteria - Bacillus -- typical rod-like bacteria - Coccobacillus is an intermediate between the two, like a shorter rod - 3 types of spirals - Vibrio (comma shaped) - Spirillum (wavy) - Spirochete (corkscrew-shaped) - Pleomorphic bacteria change from one shape to another sometimes due to environmental stressors - Arrangement: pairs, tetrads, chains - Helpful in identification - Small surface to volume ratio to allow for quick absorption of nutrients **Gram stain for bacteria** - **Sensitivity:** - Need \~ 10^5^-10^6^ CFU/ml of specimen to detect bacteria using Gram stain - A negative Gram stain does NOT rule-out infection - Could be wrong stain, sensitivity issue, viral bacteria, or wrong site - **[Challenge:]** Gram-stain helps determine org. presence more information needed to guide therapy decision (e.g. E. coli vs Pseudomonas different susceptibilities to antibiotics) **Establishing Host Colonization: Immune Evasion** **[Glycocalyx]** - Viscous, gelatinous polymer compose of polysaccharides, polypeptides, or both - **If organized and attached to cell wall = capsule** - **If unorganized and loosely attached = slime layer** - Visualized with negative stain - Used in identification -- e.g. *Salmonella enterica* serovar Typhi Vi antigen (capsule) **[Virulence factor ]** - **Prevents antibodies recognizing bacteria** - **Assists with attachment to surfaces** - Note: **antibodies cannot bind pathogen pathogen not recognized by phagocytes** **Establishing Host Colonization: Adherence** - Critical process for bacteria and viruses - Bacteria use adhesins - Viruses use **attachment proteins** - Binding to host cell **receptors** is highly specific **tropism** **Establishing Host Colonization: Biofilms** - Bacterial growth has 2 general phases: - Free-swimming (**planktonic)** - Sessile, surface associated (**biofilm**) - Biofilms are aggregates of organisms encased in a matrix consisting of proteins, carbohydrates, and nucleic acid complex structures (water channels) within the biofilms allow nutrients to be delivered to the cells - Biofilms are ubiquitous (meaning it is present anywhere and everywhere) (plaque is an example of biofilm) - They are **resistant to immune clearance, antibiotics disinfectants** stick around (in bathtubs and hospitals) - Biofilm bacteria are metabolically less active **Oxygen Requirements of Microbes** 1. Aerobes: a. Growth in ambient air (21% O2, 0.03% CO2, mostly N2) b. Require molecular O2 as terminal electron acceptor 2. Obligate Aerobes: c. Absolute requirements of O2 to be able to grow d. Usually have no fermentative pathways e. Examples = Pseudomonas, Bacillus, Mycobacterium 3. Anaerobes: f. Usually cannot grow in the presence of O2 O2 is toxic g. Use other substances as terminal electron acceptor h. Metabolism is frequently fermentative reduce organic compounds to various end products including organic acids and alcohols 4. Obligate anaerobes: i. O2 highly toxic j. Fermentative metabolism k. Examples = Actinomyces, Bacteroides, Clostridium 5. Facultative anaerobes: l. Capable of growth under both aerobic and anaerobic conditions m. Preferentially use O2 as terminal electron acceptor n. Will respire aerobically until O2 exhausted, then switch to fermentation or anaerobic respiration o. Examples = most bacteria, *Escherichia coli* **Anaerobes** - Constitute 99-99.9% of culturable flora in mucosal surfaces (e.g. oral, GI, GU) - Expect in animal/human bites, trauma, surgery - Tissue and fluids are best place for anaerobes - Suspect when organisms seen in Gram but do not grow in culture - Ideal samples: fluids, tissues in anaerobic transport media (\>1 cm^2^ does not require anaerobic media) - NEVER: superficial sites (no swabs!! -- anaerobes die d/t exposure to air), sputum, BAL, stool, vaginal; do NOT refrigerate or freeze **Antimicrobial Susceptibility Test (AST)** - - **Minimum inhibitory concentration (MIC)** - Concentration gradient of antibiotics - Lowest concentration that inhibits growth = MIC - **Zone of inhibition (ZOI)** - Concentration gradient of antibiotic in media - Measure ZOI and compare with known standards **Fundamentals of Virology** **Virus structure (influenza Example)** - Metabolically inactive on their own until they invade a cell - **Envelope** plays critical role in infection when present (lipid bilayer) - Envelope susceptible to **detergents** - **Non-enveloped** viruses persist in environment (ex: norovirus) - **Capsid** provides structure (MI -- matrix protein) - **Attachment proteins:** facilitates adhesion to cell and invasion (HA -- hemagglutinin) - Genome = **RNA** or **DNA** (segmented (-) strand RNA gene) **Virus Properties** - **Absolute dependence** on living host for reproduction (obligate intracellular parasite) - Considered **non-living** - Can cause **latent infection** whereby the virus is NOT cleared by host immune system and can **reactivate** in the future (ex. HIV, shingles) - Viral genome directs synthesis of new viral particles by usurping host cell components - Viruses can **bud** from cells (replicate so much in the cell that they spill open) or **lyse** cells - Some infections **acute or chronic** (ex, HIV) - Availability of tx uncommon usually prevention is critical (e.g. vaccines) - **Influenza virus life cycle** - Entry - Uncoating - Replication/transcription - Translation of viral proteins - Protein expression - Assembly and release **Methods of Diagnostic Virology** 1. **Virus isolation (cell culture)** a. Not all viruses are culturable in lab (needs very specific cell lines) b. Can take weeks to grow long TAT if cultured (not common) c. Some viruses may lose viability in transit to lab transport ASAP (at 4 C) or store (at -70 C if \> 72 hours from collection)! d. Yield can be limited -- depends on sample type/location (swab vs fluid aspirate; BAL vs sputum for pneumonia) 2. **Direct detection of virus** e. Electron microscope f. Pathology specimens using light microscopy g. Detection of viral antigen via direct fluorescent stain h. **Molecular techniques (PCR) viral genes** 3. **Serology (aka "immunodiagnostics"; not limited to viruses...)** i. Detection of **antibody (host)** or **antigen (virus component)** in blood j. Profile of antibody response to infection k. Used to: i. Determine **immunity (past infection OR vaccination)** to virus (measles, mumps, etc.) (**immunoglobulin G (IgG) antibodies)** ii. Recent or **acute** infection (**IgM antibodies or seroconversion)** l. Antibodies can be cross-reactive need to interpret in clinical context! A diagram of a normal life cycle Description automatically generated - - Graph 1 - Exposure to pathogen - Starts producing antibodies (ex. IgM and IgG) - IgM = early phase of response, not specific - IgM positive means infection is new - IgG = long term, specific -- indicates past infection, so the body has immunity now - Graph 2 - Lag period (seroconversion window) is shortened - Detect viral component before body's response to infection - RNA appears first = recent infection, bc IgG not present **Fundamentals of Mycology** **Overview of fungi and their infections** - Eukaryotes - Easily visible by light microscopy - Generally harmless (\