MPL 202 MIDTERM 1

• This review outlines lab errors, pathogen infection processes and their impact on hosts, and characteristics/detection of bacteria, viruses, fungi, and parasites

Path of Lab Workflow

• Within primary healthcare control: specimen collection during pre-analytical phase and patient assessment/infection control post-analytically
• Within lab control during analytical phase: specimen processing, extraction, reagent preparation, reaction, results

Errors in Total Testing Process

• Pre-analytical errors are the most common for errors to occur
• High workload and inappropriate test requests can lead to errors
• Errors can result from; incorrect order entry, patient/specimen misidentification, inappropriate timing in sample collection, with an inappropriate site, resulting in hemolyzed/clotted sample, insufficient volume, inappropriate container, handling, storage, and transportation may also contribute
• Samples not stored properly get contaminated, leading to skewed results = leads to pts being treated for something that is not actually there
• “garbage in = garbage out"
• Over 90% of diagnostic errors occur in pre- and post-analytical phases of testing
• Analytical errors account for 7-13% of errors
• Failure to detect lab QC and equipment malfunction accounts for analytical errors
• Post-analytical errors: include; failure in timely and appropriate reporting, excessive turn-around time, transcription errors, failure/delay in reporting critical values, incorrect physician interpretation

Microbes in the Environment

• Microbes are ubiquitous
• Ceiling tiles: harbor Aspergillus and other fungi
• Alcohol-based hand rub (ABHR) stations: ABHR is effective for most pathogens, not all (e.g., Clostridium difficile spores)
• High-contact surfaces (fomites): harbor MRSA, influenza, other viruses and bacteria
• Sinks: can contain water-borne pathogens (Serratia, Pseudomonas, ARO, biofilms)
• Infection Control Practice, pathogen transmission, healthcare-associated infections, epidemiology of disease and diagnostic approaches are needed to reduce spread of bacteria and disease

Phylogenetic Tree of Life

• Three domains: Bacteria, Archaea, Eucarya
• Archaea (extremophiles) have never been associated with human infection
• Eucarya includes infectious organisms like bugs

Overview of Microorganisms and Other Infectious Agents (Pathogens)

• Pathogens are microorganisms that can cause disease
• Eukaryotes are often unicellular but can be multicellular, whereas prokaryotes are unicellular
• Prions: related to mad cow disease, they are misfolded proteins that self-replicate
• Archaea: extremophiles, live in extreme temperatures, pH, and salinities
• Bacteria, viruses, viroids, and prions can cause infection
• Eukaryotic cells can be microscopic but are larger than prokaryotic cells (like bacteria), whereas Prokaryotes are larger than viruses, viroids, and prions

Basic Steps for Infection

1. Pathogen must be present
2. Susceptible host must be present
3. Entry via cuts, mucous membranes, ingestion, breathing, etc.
4. Establishment of colonization in the host via adherence, replication, and defense evasion
5. Damage to the host directly (virulence factors) or indirectly (immune response)

Portals of Entry

• Mucous membranes: respiratory, gastrointestinal, genitourinary tracts, and the placenta

• Skin: parenteral entry via bites, punctures, injections and wounds

• Most pathogens have preferred portals of entry i.e. Streptoccocus pneumoniae causes pneumonia when inhaled

• Some pathogens cause illness regardless of entry; high viral load means enough is present anywhere to cause the infection(Ebola)

• Pathogens will cause different disease depending on portal of entry i.e. Yersinia pestis (plague) can be; bubonic (flea bite) or pneumonic (inhalation)

Progress of Infection

• Exposure: contact with agent
• Colonization: persistence of microbe
• Infection: persistence with a deleterious effect on the host
• Period of communicability: when infection is transmittable
• Latency period: time between initial exposure and becoming infectious
• Incubation period: time from exposure to symptom development

Human Barriers to Infection

• Microbiota: ecological community of commensal, symbiotic, and pathogenic microorganisms, physically blocks access to mucosa below and repetitive exclusion
• Cilia: constantly forcing things away.
• Mucous: physical barrier
• Lysozyme: destroys bacterial cell walls

Pathogen Toolkit: Virulence Factors

• Virulence factors are molecules produced by pathogens that allow them to; Colonize the host, immunoevasion and immunosuppression
• Colonization has to do with; adherence (adhesions, attachment proteins).
• Destructive enzymes: destroy connective tissues via hylauronidase and destroy cells via hemolysis
• Toxins: e.g. anthrax
• Immunoevasion has to do with; Prevent antibody binding via the capsule and by the creation of a Biofilm, which will prevent phagocytosis
• Biofilms prevent WBC from performing phagocytosis

Host Damage and Infection Outcome

• Damage can be direct (toxin-mediated) or indirect via the host's immune response
• Endotoxin is part of the cell wall of some bacteria (LPS) that lyse and cause immune response, thus triggering immune cells to release cytokines in toxic concentrations. This release can also be triggered by antibiotics, so pt may ‘get worse before they get better'
• Exotoxin: enzymes secreted by bacteria that perturb aspect of normal host physiology and are generally very specific to genus/species, and highly immunogenic
• Damage to the host facilitates invasion, dissemination, and transmission; Vibrio cholera uses cholera toxin to cause diarrhea

Fundamentals of Bacteriology: Bacterial Cell Structure

• Cell membrane, surrounded by a cell wall + a capsule/slime layer
• Cytoplasm with ribosomes where and floats and vesicles may be present too
• External features include flagella and pili
• Flagellum propels bacteria cell structure
• Plasmids carry genes that give genetic advantages like antibiotic resistance

Bacteria Cell Wall

• Gram-negative: has peptidoglycan (10-20% of cell wall) and outer membrane made up of 2 membranes to counterstain. These cells Will not absorb dye, giving it a pink hue in the lab
• Gram-positive: has peptidoglycan (60-90% of cell wall) and a thick cell wall with NO outer membrane, hence staining purple, as it absorbs crystal violet dye

Bacterial Cell Wall Notes

• Gram-positive vs Gram-negative ID may direct treatment
• Bacteria have a semi-rigid cell wall exterior to the cell membrane that is responsible for maintaining the characteristic shape and protects against rupture from osmosis because it is porous, some bacterias function remains unclear, but are used by bacteriophages (viruses that infect bacteria)
• Types; spherical, spiral, rod-shaped, Pleomorphic bacteria change from one shape to another due to environmental stressors
• Arrangement: pairs, tetrads, chains, is helpful in identification
• Small surface to volume ratio allows for quick absorption of nutrients

Gram Stain for Bacteria

• Sensitivity need 105-106 CFU/ml to detect bacteria using Gram stain (a negative result doesn't necessarily mean one absence)

Establishing Host Colonization: Immune Evasion

• Glycocalyx: viscous, gelatinous polymer of polysaccharides, polypeptides that attach to cell wall (capsule), loose attachment = slime layer visualized through a negative stain

Virulence Factor

• Prevents antibodies from recognizing a pathogen
• Assists with surface attachment

Host Colonization: Adherence

• Critical for bacteria and viruses with;
  • Bacteria using adhesins
  • Viruses use attachment proteins = Binding to host cell receptors, a highly specific process known as tropism

Host Colonization: Biofilms

• Bacterial growth has 2 general phases: planktonic (free-swimming) sessile, and surface-associated.
• Biofilms= aggregates of organisms encased in a matrix of proteins, carbohydrates, and nucleic acid (water channels) that are ubiquitous and have the tendency to be resistant to immune clearance, antibiotics,
• Biofilm bacteria are metabolically less active

Oxygen Requirements of Microbes

• Aerobes: Growth in ambient air (21% O2, 0.03% CO2, mostly N2) and Require molecular O2 for terminal electron acceptor
• Obligate Aerobes: Have absolute requirements of O2 to grow, no fermentation pathways Ex: Pseudomonas, Bacillus, Mycobacterium
• Anaerobes: cannot grow in O2, use other substances as terminal electron acceptor in fermentative metabolism
• Obligate anaerobes= use fermentative metabolism Ex: Actinomyces, Bacteroides, Clostridium
• Facultative anaerobes: will grow under anaerobic conditions and they Will respire aerobically until O2 is exhausted, then switch to fermentation or anaerobic respiration Ex: Escherichia coli

Anaerobes

• Constitute 99-99.9% of culturable flora in mucosal surfaces (e.g. oral, GI, GU), expecting in animal/human bites, trauma, surgery because tissue and fluids are best place for anaerobes
• Ideal samples: fluids, tissues (>1 cm²), NO swabs and sputum since since anaerobes will die thanks to the exposure to air

Antimicrobial Susceptibility Test (AST)

• Minimum inhibitory concentration (MIC) = lowest concentration to inhibits growth
• Zone of inhibition (ZOI) = concentration of gradients of different antibiotics that will be measured to for comparison to known standards.

Fundamentals of Virology

• Viruses are metabolically inactive until inside a cell
• Envelope plays plays a critical role in infection when present, but it is susceptible to detergents
• Non-enveloped viruses: persist in environment better than enveloped viruses, an example is norovirus
• Capsid: provides structure
• Attachment proteins: facilitates adhesion to cell and invasion
• Genome can be RNA or DNA
• Virus is 100% dependent on a living host for reproduction and can cause latent reaction
• Viral genome directs the synthesis of new viral particles
• Viruses bud from cells (replicate) and some infections can be acute or chronic
• Because the availability of tx is uncommon, prevention is most important

Influenza Virus Life Cycle

• Entry, Uncoating, Replication/transcription, Translation of viral proteins, Protein expression, and Assembly and release

Methods of Diagnostic Virology

1. Virus isolation (cell culture)
   • Not all viruses are culturable and can take weeks to grow
   • Some viruses may lose viability in transit i.e move ASAP
   • Yield can be limited i.e swabs for pneumonia and BAL'S
2. Direct detection of virus
   • Through electron microscope or molecular techniques to detect viral genes
3. Serology aka "immunodiagnostics" not limited to viruses
   • to detect antibodies (host) or antigen (virus component) in their blood to show the profile of Ab's response to an to infection determine immunity that may show acute/ recent infections with IgG and IgM, it is important to note the antibodies can be cross-reactive

Fundamentals of Mycology

• Fungi Eukaryotic, visible by light microscopy, and generally harmless.
• Can cause opportunistic infection due to a immunosuppressed host
• SPECIFIC antifungal therapy is needed.

Specimen Collection and Transport

Appropriate site is needed, along with aseptic technique(avoiding bacterial contamination.

• tissue like biopsy/ scrapings/fluids NOT swabs
• Specimen should be moved sterile container, transport it to a lab to ensure that more bacteria will out grow the fungi

Types of Fungi and Features

• Single celled (yeasts) or Multi-celled (Molds) Single-celled are round/oval-shaped cells that are asxual while Multi-celled form complex structures with sexual and asxual reproduction
• Overview of Fungi and Features

Classification of Fungi and Mycosis (Fungal Infections)

• Types of fungal infections: mycoses
• They progress very slowly and are often chronic leading to difficult treatment
  • Superficial is on in skin, hair, nails, these types are commonly transmitted from human-to-human and is often immunocompetent.
  • Subcutaneous: include muscle, connective tissue, can not be transmitted or can rarely be transmitted person to person
  • Systemic: affect internal organs and cannot be transmitted person to person + it generally infects immunocompetent patients

Methods in diagnostic Mycology

• Direct detection will detect fungi in clinical samples using special stains with unique characteristics
• Culture is when you specimen into that with antibiotics to kill the bacteria, thus, we must place it in an incubation in temps from 20-37 C temp of site of infection and it can take from 5-21 days for them to grow

Fundamentals of Parasitology:

• Eukaryotes i.e Fungi and single-celled/ multi celled

General Info

• Infections may be life long/ asymptomatic such as parasite that lives on/in an host and derives nutrients are vectored to a living organism the causes diseases directly can also cause diarrheal illness/ blood stream infection
• Cause: Diarrheal illnesses and bloodstream infection >>> skin/other

Malaria

• Common in travelers returning from the tropics
• Common complaint; fever
• Incubation >21 days
• Can kill!

Parasites Important to Nursing

• Pinworms: children- primary host. transmission from Eggs (sticky), and symptoms anal Itching
• Bedbugs: breakfast, lunchtime, dinner Bugs, length is appx.4-7mm and last for the year without eating
• Lice: commonly found in kids that are 3-12 y/o, 2-3mm and survive around 48. hrs

Methods in Diagnostic Parasitology

1. Direct Detection: this depends on where the body cause is located.
2. Histopathology: biopsy the para site and tissues by finding eggs/larva or single-celled protozoa Usually done for tissue and detecting one

• Can also use larval detection directly on skin, GI tract, eyes and other sites to check trails from the parasite.