MMI 133

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MMI 133
2013-02-20 18:06:30
Part Two

Lecture 4
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  1. Viruses
    • Infectious particles, but not living cells
    • Responsible for majority of visit to GP
    • Many ways they can be transmitted from host to host (blood transfusion, mucus droplets, aerosols, fomites, water, food, vectors)
    • Must be treated using different therapeutic strategies than other microorganisms
    • Antibiotics do not work against viruses!
  2. Comparison of virus and bacteria
    • Virus Bacteria
    • 10-500nm  Bacteria: 500-~2000nm
    • Multiply only inside of cells  Most are free-living and multiply in the absence of other cells
    • Contain DNA or RNA, never both  Always contain DNA and RNA
    • Few enzymes  Contain many enzymes
    • No ribosomes or enzymes for metabolizing nutrients  Contain ribosomes and enzymes for metabolism of nutrients
  3. Virus Shapes
    • Smaller than bacteria
    • 10-500 nm diameter
    • Obligate intracellular parasites
  4. Viral Structure
    • Virion: complete infectious viral particle with nucleic acid surrounded by a protein coat
    • -Genetic material is RNA or DNA
    • -Capsid (protein coat) is comprised of capsomeres
    • Complete unit: nucleocapsid
    • Some viruses have lipid envelopes outside of nucleocapsid (these are derived fro host cell membranes, influenza virus, Herpes virus, HBV, HIV)
    • Glycoproteins can be inserted in envelopes or capsid
  5. Virion
    Complete infectious viral particle with nucleic acid surrounded by a protein coat
  6. Genetic material of Virus
    RNA or DNA
  7. Capsid
    Protein coat, is comprised of capsomeres
  8. Capsomeres
    Protein molecules that make up a protective coating on a virus. The number of capsomeres can be used to classify viruses
  9. Nucleocapsid
    The complete unit of protein coating.
  10. Lipid envelopes
    Some viruses have these outside of the nucleocapsid; that are derived from the host cell membranes
  11. Glycoproteins
    Can be inserted in envelopes or capsid
  12. Host Range
    • Different viruses can infect every life form - bacteria, fungi, plants, animals, humans
    • Usually fairly specific "host range or specificity" (bacteriophage infects only bacteria; plant viruses not infectious for humans, animals, or bacteria)
  13. Bacteriophages
    • Viruses that infect bacterial cells and can transfer new genes from one bacteria to another
    • Can be used as a tool for genetic engineering
    • Can transfer genes for production of toxin
  14. Classification
    • Viruses are grouped into families on basis of DNA/RNA composition and structure
    • (RNA viruses are known for their ability to mutate quickly)
  15. DNA viruses
    • Papilomaviridae (ie. HPV)
    • Adenoviridae (ie. adenovirus)
    • Hepadnaviridae (ie. hepatitis B virus)
    • Herpesviridae (ie. HSV-1)
    • Poxviridae (ie. smallpox)
  16. RNA viruses
    • Retroviridae (ie. HIV)
    • Favivirdae (ie. hepatitis C virus, West Nile virus)
    • Orthomyxoviridae (ie. influenza)
  17. Events occurring in viral infection
    • 1. Attachement and penetration (entry) into host cell
    • Two processes of attachment and penetration: fusion and pinocytosis
    • 2. Replication
    • Different for RNA and DNA viruses
    • 3. Assembly and release
    • Association of newly replicated RNA or DNA (nucleic acid) with new viral proteins makes the new nucleocapsid
    • If enveloped virus: envelope components are produced and inserted into the host cell plasma membrane, the viral particle then attaches to the plasma membrane and buds through
  18. Replication of Viruses
    • Different for RNA and DNA viruses
    • DNA virus: Host cell DNA polymerase is usually used directly to make more virus DNA (in host cell nucleus)
    • RNA virus: Virus must carry its own RNA polymerase enzyme to produce RNA from viral RNA (no RNA polymerase in host cells)
    • RNA retrovirus (ie. HIV) carries its own reverse transcriptase (a special type of DNA polymerase) enzyme in the virus capsid:
    • Reverse transcriptase makes ssDNA from ssRNA, then dsDNA is formed and integrates with the host DNA in the chromosome
    • Integrated viral DNA is then transcribed by the host cell polymerase and makes more virus RNA
  19. Antiviral therapies target events occurring in viral infection
    • Fuzeon - stops entry (fusion) of HIV into cells
    • Acyclovir - stops replication of herpesvirus by interfering with the viral DNA polymerase
    • HAART = highly active anti-retroviral therapy - combination of antiviral drugs used to stop replication of HIV (combination helps to prevent virus becoming resistant to drug)
    • TamiFlu (oseltamivir) - stops budding/release of influenza virus from host cell
  20. Host-virus interactions 3
    • Acute or Productive infection - virus replicates, produces many virions (influenza); host cell often killed = lytic infection
    • Latent infection - viral genome persists in host cell but does not replicate - "provirus" (herpesviruses)
    • Chronic infection - virus replicates without causing host cell lysis and can persist for long periods of tie (hepatitis C virus)
  21. Acute or Productive infection
    • Virus replicates, produced many virions (influenza)
    • Host cell often killed = lytic infection
  22. Latent infection
    Viral genome persists in host cell but does not replicate - "provirus" (herpesviruses)
  23. Chronic infection
    Virus replicates without causing host cell lysis and can persist for long periods of time (hepatitis C virus)
  24. Chickenpox: acute and latent infections
    • Acute infection: Fever, itchy (pruritic) rash (5 days)
    • Children usually have rapid onset or rash, adults a prodrome of 1-2 days (malaise, fever)
    • Rash is spread on trunk and head - less on arms and legs
    • Extent of rash usually corresponds to severity of ilness
    • Most children have 250-500 vesicular skin lesions
    • Skin vesicles always present in different stages of lesion formation
    • Latent infection: virus is present in cells but is not replicating until triggered by external factors
    • In the case of chickenpox, this is known as Shingles (Trigger? Age? Decline in immune system?)
  25. Shingles = Zoster (reactivation of latent infection)
    • Reactivation of the viral infection
    • Begins as a local skin eruption often close to the dorsal root ganglia but not exclusively there
    • Characterized by rash and pain
    • Untreated rash lasts 2-5 weeks, more confluent than chickenpox
  26. Chickenpox/Shingles = Varicella zoster virus (HHV-3)
    • Spread by airborne and direct routes
    • Vesicles in both chickenpox and shingles are infectious!
    • Incubation period to vesicles 14 days - chickenpox
    • Virus can be transmitted during the incubation period
  27. Varicella (chickenpox): Complications
    • Most common complications in immunocompromised is secondary bacterial infection
    • CNS disease: encephalitis, meningitis, myelitis
    • Hemorrhagic complications (rare): neutropenia (low number of white blood cells) and thrombocytopenia (low amount of platelets)
    • Primary varicella pneumonia is rare but very serious
  28. Shingle (zoster): complications
    • PHN (post herpetic neuralgia)
    • 25-50% of patients > 50 yrs develop PHN
    • Pain that persists for months or years
    • Can cause permanent nerve damage
    • Reactivation affecting the ophthalmic branch of the trigeminal nerve results in keratitis, blindness
    • Encephalitis in up to 0.5% of cases
  29. Some viruses can "transform" normal host cells to cancer cells
    • These viruses are "oncogenic" viruses
    • Not all "transformed" cells become cancerous eg. wart virus: often benign tumor
    • Cancer-producing viruses can be RNA or DNA viruses
  30. Viral Diagnosis: Cell Cultures
    • Patient specimens added to cultured cells and after a period for growth, observed for CPE (cytopathic effects) on the host cells
    • Different viruses grow in different types of cells - must have a battery of cell lines
    • Labour intensive, slow (dependent on viral growth rates)
  31. Viral diagnosis:
    • Influenza A and B in culture
    • FA stain (fluorescent antibody)
    • Often used as "DFA" (direct fluorescent antibody) test for viral pathogens (quick + easy)
  32. Sequence of events in smallpox infections:
    • Smallpox enters through mucosal membranes of the respiratory system - then enters lymphoid tissue (droplet transmission, viral particles on objects used for bioterrorism against Native North Americans)
    • Viremia (viruses in bloodstream) and spread of virus through body
    • Clinical disease, development of lesions: popular 1-4 days, vesicular 1-4 days, pustular 2-6 days, crusts fall off 2-4 weeks after first sign of lesion
  33. Difference between Chickenpox and Smallpox
    • Chickenpox - lesions are in different stages
    • Smallpox - all lesions are in the same stage of development
  34. Prions
    • Defined in 1982 by Stanley Prusiner
    • Protein misfolding disease
    • Self protein becomes changed and nonfunctional
    • Prions are misfolded proteins that act as infectious agents in susceptible exposed animals
    • Normal protein     Misfolded protein
  35. Take home messages about viruses and prions
    • Viruses are different from prokaryotes and eukaryotes (know differences)
    • Viruses are classified based on DNA/RNA composition and structure
    • Viruses interact with the host in different ways to cause acute, latent or chronic infections
    • Prions are misfolded proteins that act as infectious agents
  36. Antiseptic
    Disinfectant used on the skin
  37. Aseptic technique
    Use of methods to exclude microorganisms
  38. Bactericidal
    Kills bacteria
  39. Bacteriostatic
    Inhibits growth of bacteria, doesn't kill
  40. Disinfectant
    Chemical used to destroy many microorganisms and viruses
  41. Fungicide
    Kills fungi
  42. Pasteurization
    Brief heat treatment used to reduce the numbers of organisms and to kill pathogenic organisms
  43. Sanitization
    Reduction of the # of organisms to a level that meets public health standards
  44. Sterilization
    Destruction of all forms of microorganisms, including spores
  45. Viricide
    Inactivates viruses
  46. Physical Methods for microbial control
    • Ventilation: "Air locks"
    • Positive pressure room: air flows only from the patient room to the corridor - not from the corridor in to the room
    • Negative pressure room: air flows from the corridor in, not from the patient room out.
  47. HEPA filter
    High-efficiency particulate air filter
  48. 7 physical means of controlling microbial growth
    • 1. Heat
    • 2. Filtration
    • 3. Cold
    • 4. High Pressure
    • 5. Desiccation (drying)
    • 6. Osmotic pressure
    • 7. Radiation
  49. Sterilization may be achieved by:
    • a. heat: hot air (160 - 180oC) for 1-2 hrs;
    •              autoclaving (moist heat): 121oC, 15 psi, 15' will kill C. botulinum spores
    • -test for effective sterilization: "spore test" using Bacillus stearothermophilus spores
    • b. irradiation (gamma or UV)
    • c. filtration
    • d. chemicals
    • - most important consideration that determines the efficiency of sterilization is whether or not the object to be sterilized in free of organic matter (ie. Blood, fecal material, tissue)
  50. "spore test"
    • Based on testing the ability of the autoclaving procedure to totally inactivate bacterial spores so that they will not be able to grow after a successful autoclaving
    • Ampule containing nutrient media and spores of a non-pathogenic bacteria are run with the object to be sterilized in the autoclave. After the process, the ampule is incubated at 45o C to test for growth
  51. Chemical means of controlling growth
    • 1. Hand soap: surface active agents
    • 2. Phenols - god disinfectants, remain active in presence of organic material (Lysol)
    • 3. Bisphenol - antiseptic (Phisohex)
    • 4. Biguanides - antiseptic (Chlorhexidine)
    • 5. Quaternary ammounium compounds: (Cepacol)
    • 6. Alcohols - isopropyl/ethanol
    • 7. Heavy metals - silver, mercury
    • 8. Halogens - chlorine, iodine (can sterilize if used well)
    • 9. Aldehydes: sterilize! (glutaraldehyde or Cidex)
    • 10. Ethylene oxide: gas that sterilizes
    • 11. Peroxygens: strong oxidizer, sterilizes
  52. Chemicals that Sterilize
    • Halogens - Chlorine, iodine (can sterilize if used well)
    • Aldehydes: sterilize! (glutaraldehyde or Cidex)
    • Ethylene oxide: gas that sterilizes
    • Peroxygens: strong oxidizer, sterilizes
  53. Most used and useful "disinfectants" in everyday health care situations
    • Halogens: iodine and chlorine
    • -Iodine: available as tincture (sol'n in alcohol) or as an iodaphor (comb. of iodine and organic molecule which releases iodine slowly) eg. Betadine
    • Chlorine: strong oxidizing agent eg. Chlorox
    • Note that halogens can sterilize if appropriate amount of active chemical is used and prolonged time of exposure
  54. Alcohols
    • Kill bacteria and fungi but not endospores and non-enveloped viruses
    • Mechanism is protein denaturation and disruption of the lipid membranes
    • Used for skin "degerming"
    • Not good for treating wounds, as they cause a coagulation of proteins, creating an environment where the bacteria can grow
    • Optimal conc. ethanol = 70% (denaturation needs water to work)
    • Isopropanol = rubbing alcohol - is better than ethanol; does not evaporate as fast
  55. Relative resistance of microorganism to chemical agents
    Least suspectible -> Most suspectible
    • Prion
    • Endospores
    • Mycobacteria
    • Cysts of vegetative protozoa
    • Vegatative protozoa
    • Gram negative bacteria
    • Fungi
    • Naked viruses
    • Gram positive bacteria
    • Enveloped viruses
  56. Sterilization
    Inactivation of spores
  57. Antisepsis
    For skin
  58. Disinfection
    For surfaces
  59. Weapons against infection
    • Antibiotics
    • Antiviral agents
    • Antifungal agents
    • Antiparasitic agents
    • Some immunoactive substances (interferon)
    • Maintenance of immune integrity, hygiene
  60. No antibiotic will be effective if used too late in the infection
    • Too many bacteria
    • Too much tissue damage
    • Formation of walled-off abscesses that can't be penetrated by antibiotics
    • Poor absorption of drug
  61. SULFA
    1st synthetic antimicrobial substance to act selectively on bacteria
  62. Antibiotics don't kill human cells:
    • because they attack the differences:
    • Prokaryotes have cell wall, different ribosomes (70s), may need folic acid, so we produce antibiotics that have selective effect on bacteria, not humans.
  63. Bacteria mutation
    Production of beta-lactamase: inactivates penicillin
  64. Semi-syntetic penicillin
    We reacted to bacteria mutation by synthesizing this with a structure where the active portion of the antibiotic (beta-lactam ring) was protected from the enzymes (added a side chain to the molecule)
  65. Bacteria's reaction to the semi-synthetic penicillin
    • Mutated again, changing the binding sites on the bacterium to produce a variant that was resistant tot he methicillin
    • PBPs are enzymes in the cell wall for peptidoglycan synthesis but they also bind penicillin
    • (Penicillin Binding Protein (PBP) 2 mutates to PBP 2a - has reduced affinity for the penicillin, so reduced uptake into cell wall and penicillin cannot inhibit the crow-linking of peptidogylcan
    • eg. MRSA = methicillin resistant S. aureus
  66. Bacteriostatic antibiotics
    • Stop the replication of bacteria
    • Do not kill the bacteria already present
    • eg. erythromycin
    • (most can be bacteriostatic or bactericidal dependent on concentration and situation)
  67. Bactericidal antiboitcs
    • Kill the bacteria
    • Stop bacterial metabolism
    • eg Aminoglycosides like gentamicin
    • (most can be bacteriostatic or bactericidal dependent on concentration and situation)
  68. Specific antibiotics
    Inhibt gram - or gram + organisms or certain bacterial species
  69. Broad-spectrum antibiotics
    inhibit both Gram - and Gram + organisms: wide variety
  70. Major Classes of Antimicrobials
    • 1. Agents that cause inhibition of cell wall synthesis (penicillins, cephalosporins, vancomycin)
    • 2. Agents that cause inhibition of protein synthesis (aminoglycosides, macrolides, tetracyclines)
    • 3. Agents that cause injury to the plasma membrane (polymixin B (topical))
    • 4. Agents that inhibit nucleic acid synthesis (quinolones, rifamycins, antivirals like acyclovir)
    • 5. Agents that inhibit the synthesis of essential metabolites (sulfa and trimethoprim)
  71. Drug Effux pump
    Some bacteria actively pump antibiotics out of the cell - eg. Pseudomonas species pumps out tetracyclines and quinolines
  72. Problem with anti-viral agents
    Mutation of viruses - become resistant to antivirals (especially RNA viruses)
  73. Quinine and derivatives for malaria
    • Are anti-protozoan and anti-helminthic drugs
    • Problem: increasing resistance of malaria to drugs
    • Anti-helminthis drugs work in novel ways: eg. niclosamide for tapeworms (inhibition of ATP production)
    • praziquantel: schistosoma, tapeworms (alters permeability of membranes, exposes surface antigens to the mmune system)
    • mebendazole: ascrais, pinworms, trichuris (disrupts microtubules - reduces worm activity)
  74. 3 mechanisms for Foreign DNA uptake
    • Transformation requires dead bacteria
    • Transduction viral delivery
    • Conjugation sex pilus
    • transfer the plasmids.
  75. 3 reason bugs become resistant
    • overuse
    • incomplete treatment regimens
    • Inappropriate treatment
    • all bugs don't need drugs!!
  76. Superbugs
    Bacteria that can't be controlled by antibiotics
  77. ESBL (Extended Spectrum Beta-Lactamase)
    • Gram - bacteria (enterobacteriaeae) genetic mutations, many genes eg. recent reports of NDM-1
    • Increasing problem in ICU, extended care
  78. MIC
    • Minimum Inhibitory Concentration
    • Minimum amount of the antimicrobial needed to inhibit (but not kill) the microorganism
  79. Resistant Microbes
    • MRSA (Methicilin resistant Staphylococcus aureaus) (PBP 2 is mutated to PBP 2a)
    • VRE (Vancomycin resistant enterococci) (developed after use of "avoparcin" in animal feed)
    • ESBL (Extended Spectrum Beta-Lactamase) (increasing problem in ICU, extended care)
    • Pseudomonas aeruginosa (can live in liquid soaps, needs very few nutrients, can infect many body sites and very common in burn patients and patients with cystic fibrosis)
    • Streptococcus pneumonia (increasing resistance to penicillin due to mutations in PBP, common cause of CAPD)
  80. Disk Diffusion Test (or Kirby Bauer test) for Determination of Antimicrobial Sensitivity
    • Routine test used in labs, primarily for rapidly growing bacteria as the antibiotics on the discs will deteriorate with time.
    • Three categories of sensitivity are defined
    • S = sensitive or susceptible
    • I = intermediate
    • R = resistant
  81. Drugs with "I" should not normally be used unless:
    • The infection is an urinary tract infection.
    • In this case, a drug may act effectively as the kidney concentrates antibiotics and the level in the urine may be 100 x greater tan in the blood
  82. Monitoring of antimicrobials in blood
    • Attain effective levels (over MIC)
    • Prevent toxic side effects
    • Ascertain dosing intervals