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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!
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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
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Virus Shapes
- Smaller than bacteria
- 10-500 nm diameter
- Obligate intracellular parasites
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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
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Virion
Complete infectious viral particle with nucleic acid surrounded by a protein coat
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Genetic material of Virus
RNA or DNA
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Capsid
Protein coat, is comprised of capsomeres
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Capsomeres
Protein molecules that make up a protective coating on a virus. The number of capsomeres can be used to classify viruses
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Nucleocapsid
The complete unit of protein coating.
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Lipid envelopes
Some viruses have these outside of the nucleocapsid; that are derived from the host cell membranes
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Glycoproteins
Can be inserted in envelopes or capsid
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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)
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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
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Classification
- Viruses are grouped into families on basis of DNA/RNA composition and structure
- (RNA viruses are known for their ability to mutate quickly)
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DNA viruses
- Papilomaviridae (ie. HPV)
- Adenoviridae (ie. adenovirus)
- Hepadnaviridae (ie. hepatitis B virus)
- Herpesviridae (ie. HSV-1)
- Poxviridae (ie. smallpox)
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RNA viruses
- Retroviridae (ie. HIV)
- Favivirdae (ie. hepatitis C virus, West Nile virus)
- Orthomyxoviridae (ie. influenza)
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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
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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
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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
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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)
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Acute or Productive infection
- Virus replicates, produced many virions (influenza)
- Host cell often killed = lytic infection
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Latent infection
Viral genome persists in host cell but does not replicate - "provirus" (herpesviruses)
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Chronic infection
Virus replicates without causing host cell lysis and can persist for long periods of time (hepatitis C virus)
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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?)
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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
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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
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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
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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
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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
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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)
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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)
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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
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Difference between Chickenpox and Smallpox
- Chickenpox - lesions are in different stages
- Smallpox - all lesions are in the same stage of development
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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
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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
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Antiseptic
Disinfectant used on the skin
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Aseptic technique
Use of methods to exclude microorganisms
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Bactericidal
Kills bacteria
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Bacteriostatic
Inhibits growth of bacteria, doesn't kill
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Disinfectant
Chemical used to destroy many microorganisms and viruses
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Pasteurization
Brief heat treatment used to reduce the numbers of organisms and to kill pathogenic organisms
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Sanitization
Reduction of the # of organisms to a level that meets public health standards
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Sterilization
Destruction of all forms of microorganisms, including spores
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Viricide
Inactivates viruses
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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.
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HEPA filter
High-efficiency particulate air filter
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7 physical means of controlling microbial growth
- 1. Heat
- 2. Filtration
- 3. Cold
- 4. High Pressure
- 5. Desiccation (drying)
- 6. Osmotic pressure
- 7. Radiation
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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)
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"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
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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
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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
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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
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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
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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
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Sterilization
Inactivation of spores
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Disinfection
For surfaces
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Weapons against infection
- Antibiotics
- Antiviral agents
- Antifungal agents
- Antiparasitic agents
- Some immunoactive substances (interferon)
- Maintenance of immune integrity, hygiene
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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
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SULFA
1st synthetic antimicrobial substance to act selectively on bacteria
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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.
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Bacteria mutation
Production of beta-lactamase: inactivates penicillin
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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)
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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
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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)
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Bactericidal antiboitcs
- Kill the bacteria
- Stop bacterial metabolism
- eg Aminoglycosides like gentamicin
- (most can be bacteriostatic or bactericidal dependent on concentration and situation)
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Specific antibiotics
Inhibt gram - or gram + organisms or certain bacterial species
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Broad-spectrum antibiotics
inhibit both Gram - and Gram + organisms: wide variety
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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)
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Drug Effux pump
Some bacteria actively pump antibiotics out of the cell - eg. Pseudomonas species pumps out tetracyclines and quinolines
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Problem with anti-viral agents
Mutation of viruses - become resistant to antivirals (especially RNA viruses)
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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)
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3 mechanisms for Foreign DNA uptake
- Transformation requires dead bacteria
- Transduction viral delivery
- Conjugation sex pilus
- transfer the plasmids.
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3 reason bugs become resistant
- overuse
- incomplete treatment regimens
- Inappropriate treatment
- all bugs don't need drugs!!
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Superbugs
Bacteria that can't be controlled by antibiotics
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ESBL (Extended Spectrum Beta-Lactamase)
- Gram - bacteria (enterobacteriaeae) genetic mutations, many genes eg. recent reports of NDM-1
- Increasing problem in ICU, extended care
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MIC
- Minimum Inhibitory Concentration
- Minimum amount of the antimicrobial needed to inhibit (but not kill) the microorganism
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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)
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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
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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
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Monitoring of antimicrobials in blood
- Attain effective levels (over MIC)
- Prevent toxic side effects
- Ascertain dosing intervals
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