:) Controlling Contamination Flashcards
1
Q
Sterility of Medical Products
A
Non-sterile but limits number of contaminants and specific microbes, e.g. E. coli:
- Oral
- Rectal
- Topical
- Inhaled
Sterile:
- Ophtalmic
- Parenteral
2
Q
Sterility of Medical Products - What 5 factors are risks dependant on?
A
Risks may depend on:
1. Aqueous/non-aqueous
2. Use of product; hazard dependent route
3. Nature of product: may support microbial growth, presence of preservatives
4. Intended recipient: risk differs for e.g. neonates vs. healthy adult
5. Presence of disease, wounds, organ damage
3
Q
Resistance to sterilising agents and processes - Least to most resistant (6):
A
- Least: Multicellular organisms
- Vegetative bacteria and fungi, algae, protozoa, large viruses
- Fungal spores
- Small viruses
- Bacterial endospores
- Most: Prions
4
Q
List the 5 Steps in Biofilm formation:
A
Biofilm formation:
1. Initial attachment
2. Irreversible attachment
3. Microcolony formation
4. Maturation
5. Dispersal
5
Q
List 5 Reasons biofilms are medically relevant
A
- Majority of infections involve cells growing in biofilms (e.g. wound infections, endocarditis, otitis media, cystic fibrosis)
- Indwelling medical devices (e.g. catheters)
- Growth on surfaces in e.g hospitals
- Air/water handling systems
- Biological liquid systems (e.g. dialysis equipment)
6
Q
List 4 facts about Bacterial Sporulation
A
- Survival strategy when nutrients become exhausted
- Mechanism used by some Gram-positive bacteria
- Spores are very resistant to heat, radiation, desiccation and chemical agents
- Can survive for many years
7
Q
Bacterial Sporulation 4 step process:
A
- Asymmetric cell division occurs, producing a forespore, a region where the cell containing chromosome is engulfed in peptidoglycan. 2. This results in the formation of a peptidoglycan cortex, which then gathers a protein spore coat.
- The cell undergoes lysis, releasing the spore
- The spore germinates into another bacterial cell.
8
Q
What are the three types of Fungi
A
Fungi 3 types:
- yeasts
- multicellular filamentous moulds
- macroscopic filamentous fungi
9
Q
Give three examples of pathogenic fungi, and a non-pathogenic fungi
A
Examples of pathogenic fungi:
1. Candida spp. (e.g. C. albicans) causing oral/vaginal thrush, nail infections, nappy rash, ocular candidiasis
2. Aspergillus fumigatus causing pulmonary infections
3. Dermatophytes - fungi that colonise skin, nails or hair
4. Non-pathogenic fungi e.g. Penicillium producing penicillins
10
Q
List three other rare contaminations of Medicinal Products
A
Other rare contaminations (Virus, Protozoa, Prions):
1. Viruses – found in some animal or human derived products
2. Protozoa – eg improperly sterilized contact lenses
3. Prions – blood products or medical equipment after surgery of patients with CJD; very difficult to remove (heat & chemical resistant)
11
Q
Describe the Consequences of Microbial contamination: Health hazard
A
Health hazard
- Organism or toxins can cause disease
- Depends on patient, product, route of administration
12
Q
Describe the Consequences of Microbial contamination: Spoilage
A
Spoilage
-Therapeutic breakdown of actives, or another component of formulation
- Might lose aesthetic appeal causing Noncompliance
- Financial implications
13
Q
Describe Sterile Products
A
Sterile e.g. injectables, ophthalmic preps.
- single use sterile packs OR aseptic manufacture in a clean room
- potentially exposed to micro-organisms during storage
14
Q
Describe Non-sterile products
A
Non-sterile e.g. oral, topical, rectal, vaginal, inhaled
- (contain micro-organisms; DON’T affect product quality
- potentially exposed to micro-organisms during storage and use
15
Q
What are 3 ideal properties of preservatives?
A
- decrease the risk of microbial contamination throughout product shelf life
- integral to the formulation design
- not included to counteract a poor manufacturing process
16
Q
Organic acids - 7 Facts
A
Organic acids - e.g. benzoic acid:
1. limited by pH dependence;
2. work at pH values up to pKa
3. Active at [low]
4. Non toxic, cheap, stable.
5. Reasonable spectrum of activity: bactericidal, fungicidal (not sporicidal)
6. Physically undetectable
7. Long acting (not fast acting)
17
Q
Parabens - 8 Facts
A
Parabens e.g. methyl paraben:
1. overcome pH dependence of organic acids
2. used in combination
3. BUT not very soluble, slow rate of kill
4. Active at [low]
5. Non toxic, cheap, stable.
6. Reasonable spectrum of activity: bactericidal, fungicidal (not sporicidal)
7. Physically undetectable
8. Long acting (not fast acting)
18
Q
Describe the Evaluation of Preservatives - The Challenge test
A
Challenge test: Assess activity with product in final container.
- Inoculate preserved product with 10^5-10^6 test organisms /ml or /gram of product
- Incubate at specific temp & sample over 28 days
- Assess viability by VC
- Preservative adequate if decrease in viability meets acceptance criteria.
19
Q
Describe the Suspension test
A
Simpler form of challenge test – early stage development of biocides:
- Microorganism added to antimicrobial in (aq) solution (106 CFU/mL), samples are taken at specific times, & inoculated in broth with a neutralizer (inactivator broth)
- Check for growth/no growth (i.e. does the broth become cloudy) OR serial dilution & plating to count survival
20
Q
5 Factors affecting choice of any preservative
A
- Intended application
- No. & type micro-orgs present
- Safety, stability & Cost
- Micro-environment
- Properties of chemical agent
21
Q
Intended application
A
Product type – sterile vs. non sterile
Administration route – toxicity of preservative
Moisture content
Susceptibility to attack
22
Q
What 3 ways do microorganisms effect the use of preservatives?
A
- What are the most likely contaminating organisms?
e.g. Oral products:
Bacteria: E. coli, S. aureus, P. aeruginosa
Fungi: Candida sp - How many?
- Reduce potential for contamination through packaging
- Shelf-life
- Where will it be stored and for how long?
23
Q
How does Safety, stability & cost effect preservatives?
A
Safety:
- safe to handle during manufacture / packaging
- non-toxic in relation to route of administration
Stability:
- unaffected by products in formulation
- effective over pH range
- effective over shelf life
Cost effective:
- must be relatively low cost
24
Q
How does the Micro-environment effect preservatives?
A
Availability of preservative
- Interaction with formulation and/or with container?
- Permeation through the container & evaporation?
- Partitioning into oily phase? (e.g. parabens)
Moisture
- Products with large amounts of water are more at risk
- Aim to reduce moisture content
Storage temp
- Affects activity / stability of preservative
- Affects levels of contamination
- Consider fluctuations
25
How do the Physiochemical properties of product effect preservatives?
pH of the product
- Affects potency through ionisation
e.g. Organic acids are inactive when ionised
- Affects survival & growth of organism
[Preservative]
- toxicity or very [low] required can make preservative inappropriate
26
Define: Sterilisation (Old and New), and why the change occured
Free from all viable forms of life.
But Sterilisation is not all or nothing:
- Old (wrong) concept: once a specific temp. and time is reached & maintained, all microorganisms are killed.
- Instead: sterilisation proceeds like a first-order chemical reaction; a population sterilised decreases exponentially - but never reaches zero
The change occurred because:
1. Negative state is hard to prove
2. Can we detect all viable forms of life?
3. Sterilisation process efficacy organism dependant
27
Describe the Practical approach of sterility use, SAL & PNSU
Practical approach:
Instead of using absolute sterility, probabilities are being used:
- Sterility Assurance Level (SAL)
- Probability of a Non-Sterile Unit (PNSU)
Important is how many bacteria are in the product before sterilisation (=bioburden) & acceptable level of contamination.
28
How does long sterilisation times effects sterility assurance, & what is the minimum PNSU standard?
Long sterilisation times will be effective in achieving high level of sterility assurance, but:
- drug may degrade
- process is time consuming
- expensive
Most products the minimum standard is a PNSU of 1 in 10^6
29
Describe the 3 step sterilisation process:
1. Destruction - e.g. flaming, chemical oxidation
- not practical for pharmaceutical products
2. Killing/inactivation
- used in most official methods
- may not remove endotoxins
3. Removing
- filtration, centrifugation
30
What are the 5 processes of sterilisation in the British Pharmacopeia?
1. steam sterilisation
2. dry heat
3. ionising radiation
4. gaseous sterilisation
5. filtration
31
Moist heat sterilisation - 3 facts and its uses
1. Involves steam at 121-134 degrees celsius
2. Very effective
3. Widespread application
Used for dressings, sheets, equipment, containers, aqueous injections, ophthalmic preparations, contaminated waste materials etc.
32
Dry heat sterilisation - 2 facts and its uses
1. Usually in the range of 160-180oC
2. Less effective than moist heat sterilisation
Used for glassware, metal surgical instruments, non-aqueous thermostable liquids, thermostable powders
33
Radiation - 2 facts and its uses
1. Sterilisation using gamma rays, accelerated electrons, X rays or UV
2. Alternative method for heat-sensitive products
Mainly for articles in dried state, e.g. surgical instruments, sutures, plastic syringes, dry pharmaceutical products etc.
34
Gaseous sterilisation - 2 facts and its uses
- Uses ethylene oxide or formaldehyde
- Not same sterility assurance as heat - only used for heat sensitive items
Mainly for reusable surgical instruments, medical/diagnostic equipment, surface sterilisation of powders
35
Filtration - 3 facts and its uses
- For sterilisation: 0.2-0.22µm filters used
- Only process that removes microorganisms
- Used for removal of particulates from gases and liquids
For heat sensitive injections, ophthalmic preparations, biological products, and air and other gases for supply to aseptic areas
36
Sterilisation processes - 3 facts, and describe both Terminal Sterilisation & Aseptic processing
1. Compromise between antimicrobial activity & product stability
2. Limiting factors include cost, nature of product & nature of microbial contamination
3. Process needs to be validated using suitable test organism, often on "worst case scenario" e.g. bacterial spores
If possible, use terminal sterilisation: product sterilised in final (closed) container
Otherwise, use aseptic processing: e.g. pre-sterilised components to assemble the product, requires a clean room
37
What 4 factors determines which sterilisation process is used?
Which sterilisation process to use?
1. stability & nature of product
2. scale of production
3. type & level of contamination
4. cost
38
What 3 actions are taken if the sterilisation process degrades the product?
1. adapt (or use different) sterilisation process
2. reformulate product to increase stability
3. reduce initial bioburden
39
How do Survivor curves standardly behave?
Normally, cells lose viability exponentially, i.e. microbial inactivation is analogous to first-order chemical reaction
40
What does initial clumping mean in Survivor curves?
Initial shoulder:
- clumping: each cell in cluster needs to be "hit" before CFU goes from 1 to 0
- repair mechanisms in the cell: some cells need to be “hit” twice to result in death
41
What does initial exponential, then flatter mean in Survivor curves?
Tailing curve: initial exponential, then flatter
- mixed population: different bacteria present, different resistances
- protective effects: lysis of cells protects surviving cells (e.g. pH changes)
42
What does an initial hump, then exponential mean in Survivor curves?
Activated survival curve: initial hump, but then exponential
- germination of spores upon heat stimulation
43
Describe the Expression of resistance: D value
D value (decimal reduction time):
- time needed to reduce population by 90%
- can be used both for heat & radiation
- refers to specific temperature or radiation dose
- the higher the D value, the more resistant a bacterium
D value calculation:
(t2-t1)/ (log(N1)-Log(N2)) = D value
With radiation, time is replaced with dose.
t - time (min), N-surviving fraction
44
Describe the Expression of resistance: Z value
- The Z value is the temp increase that will reduce the D value by 90%
- The Z value is the temp increase that will result in the bacteria being killed 10 times faster.
(T2-T1)/(logD1-logD2) = z value
T-Temp, D-Time
45
Define: k, the IF (Inactivation factor) & the F0
k - inactivation rate constant (k=2.303/D)
Inactivation factor (IF) - measure of total microbial inactivation
F0 - equivalent time of moist sterilisation at 121C, with a Z value of 10oC
46
Define: PNSU & SAL
PNSU: probability of a non-sterile unit
SAL: sterility assurance level
47
Autoclaving - 3 facts
1. Moist heat with temp >100oC can only be achieved under pressure
2. Performed in autoclave - similar to domestic pressure cooker, but better facilities for control
3. Organisms killed by combination of:
- Temperature
- Hydration
- Time
- Pressure has no direct lethal effect
48
Properties of steam within a closed container:
In closed container:
PV/T=constant
P-Pressure V-volume T-temperature
At constant volume, pressure varies with T
49
Describe: Superheated Steam
Superheated steam is water in the vapour phase & behaves like a gas. If the temperature decreases, the pressure decreases.
Superheated steam has both sensible & latent heat, but in absence of condensation will release only sensible heat.
50
Describe: Supersaturated Steam
Supersaturated steam or wet steam: (l), made up of small droplets of water held in suspension by convection currents.
Supersaturated steam has no latent heat, will release only sensible heat.
51
Describe: Dry saturated steam
Dry saturated steam:
- On the phase boundary.
- If you lower the temperature it will condense.
- Autoclaving uses this as it prevent excess moisture compromising the sterilisation process.
- Dry saturated steam will release both sensible and latent heat – when it makes contacts with an object, both sensible & latent heat is released, the latent heat release is required for autoclaving.
52
What 3 factors mean only dry saturated steam is suitable for sterilisation?
Factors playing a role in this:
1. Release of heat
2. Hydration
3. Penetration
53
Describe: Sensible heat
Sensible heat: The energy required for the change in physical state, causes temp change
54
Describe: Latent heat
Latent heat: Released when change in physical state occurs, no temp change occurs
55
Describe the process of Hydration: Dry saturated steam
Dry saturated uses a moist heat reaction on a spore, using rapid heat transfer to inactivate the spore, but the hydration reduces the effectiveness of his actions.
56
Describe the process of Hydration: Superheated steam
Superheated steam uses a dry heat reaction on a spore, using oxidation to rapidly hydrolyse the spore to become inactive. This process is more effective than dry saturated steam.
57
Penetration: Dry saturated steam
Dry saturated steam uses condensation of the spore to create a local vacuum, drawing in more steam for rapid penetration.
58
Penetration: Superheated steam
Superheated steam doesn’t cause condensation or a vacuum, resulting in poor penetration.
59
Bench autoclave - 6 facts
1. Easy to use
2. Works well for small number of items
3. Doesn't superheat as water is within vessel
4. if temp increases, more steam is produced
5. if both temp & pressure increase, steam remains on boundary phase
6. Inefficient if vessel gets too large: heat loss & ineffective large steam volume generation
60
Gravity displacement autoclave - 6 facts
Gravity displacement autoclave:
1. steam added from top, air is removed at the bottom
2. for laboratory media, water, pharmaceutical products, waste, non-porous items
3. unsuitable for porous loads: air may be entrapped in packaging or material
4. Uses separate steam generator
5. Cycle times shorter as steam immediately available
6. Often have a heated outer jacket for heat distribution & cooling
61
Porous load autoclave - 5 facts
Porous load autoclave (vacuum assisted):
1. fitted with vacuum pump to remove air before adding steam
2. for e.g. dressings, wrapped or hollow instruments
3. Use separate steam generator
4. Cycle times shorter as steam immediately available
5. Often have a heated outer jacket for heat distribution & cooling
62
3 Stages of operation - Pourus load autoclave
1. Air removal and steam admission:
- downward air displacement or evacuation; porous load pulsed evacuation/steam admission to ensure removal of air which reduces efficiency
2. Heating up & exposure
- sterilising time starts when operating temp. is reached
3. Drying & cooling
- porous loads may get damp; drying by vacuum &/or heat from outer jacket
- cooling necessary to shorten cycle; e.g. pumping water through jacket or spray-cooling
63
Describe: Air ballasting (autoclaving with pressure support)
- sterile fluids packaged in flexible plastic containers (e.g. IV -fluids)
- pressure in airspace may lead to bursting
- to prevent, some air may be retained to create overpressure in specifically designed autoclaves
- to prevent layering of air, such autoclaves may require fan or spraying mechanism to mix air & steam
64
Sterilisation problems: Superheated steam
Superheated steam:
1. too dry steam supply
2. overheating steam jacket
3. excessive pressure reduction (steam produced under higher pressure; reduction of pressure results in generation of heat)
4. hydration of over-dried cotton fabrics (re-hydration is exothermic process)
65
Sterilisation process: Air in steriliser
Air in steriliser:
With steam/air mixtures, total pressure is equal to sum of partial pressures, which are proportional to relative amount of steam & air present
Ptotal=Pwater+Pair
66
Describe: Layering
Layering:
Density of air = ~2 x density of steam
Air forms layer in any container in upright position, this is used to remove air from the autoclave.
67
Describe: Unjacketed bench autoclave
Unjacketed bench autoclave
- Controlled by pressure only
- Air leads to lower temp
Example: at 20psi, T = 125oC if steam is saturated
50% steam, 50% air: Ptotal = Pwater + Pair
20 = 10 + 10
At 10psi, temp. of dry saturated steam is only 115oC, while pressure gauge (at 20psi) suggests 125oC
68
Describe: Jacketed bench autoclave
Jacketed autoclave
- Both pressure & temp controlled
- Air can lead to superheating
Example: run autoclave at 121C, 15psi
80% steam, 20% air: Ptotal = Pwater + Pair
15 = 12 + 3
12 psi is equivalent to dry saturated steam at 118oC
But temperature is 121oC, thus 3oC superheating
69
Describe: Dry heat sterilisation
- Inactivation is principally by oxidation
- Oxidation is less effective than hydrolysis in inactivating bacterial spores - requires higher temp for longer time
- Official processes have smaller margin for error than moist heat sterilisation; conditions carefully controlled
- Dry heat used for sterilising items that can’t be sterilised other methods
70
4 Uses of dry heat sterilisation and their examples
1. Substances that can’t be subjected to moisture e.g. Water soluble powders (thermostable)
2. Materials not penetrable by steam e.g. Oils, dry items enclosed in sealed container etc.
3. Glassware e.g. Glass vessels, syringes etc.
4. Metal surgical instruments e.g. Scalpels, forceps etc
71
List 4 Disadvantages of dry heat sterilisation
1. may take long time to heat up
2. temp must be higher & longer than for moist heat sterilisation
3. some objects may oxidise at high temp
4. large temp variations can be found in a load
72
Adv & Dis: Sterilisation by gas
Adv:
- alternative for materials sensitive to heat or radiation
- some processes will even work at room temp
Dis:
- slow: unsuitable for high throughput sterilisation
- toxic - complex equipment, safe working protocols
- gas may be absorbed - requires addition of more gas during process
73
Adv & Dis: Ethylene Oxide (Gas sterilisation)
Adv:
- broad spectrum, non-selective
- bactericidal
- can act at room temp.
Dis:
- Expensive
- difficult, potentially dangerous – toxic
- need to store products after sterilisation in safe area while gas disperses
- requires carefully controlled humidity
- possibly explosive, [dependant]
74
Gas sterilisation - Formaldehyde (method of use)
- LTSF (low temperature steam-formaldehyde) operates with sub-atmospheric pressure steam.
- air removed, steam admitted to heat load
- release of formaldehyde by vaporisation from formalin
- after sterilisation formaldehyde vapour expelled with steam flushing, followed by drying
75
Adv & Dis: Formaldehyde (Gas sterilisation)
Adv:
- broad spectrum, non-selective
- bactericidal
- not flammable or explosive
Dis:
- toxic
- low penetration power; needs vacuum pulsing
- slow acting
- may leave residue of polymers
- need to store products after sterilisation in safe area while gas leaches out
76
5 Adv of ethylene oxide over LTSF
Adv of ethylene oxide over LTSF
1. Wider regulatory acceptance
2. Better penetration (plastics/rubber)
3. Shorter cycle times
4. Can be used at RT
5. Low incidence of product deterioration
77
2 Adv of LTSF over ethylene oxide:
Adv of LTSF over ethylene oxide:
1. Less hazardous (not flammable, readily detected by smell)
2. Gas obtained from (aq) solution (formalin) instead of gas cylinders
78
Radiation sterilisation - the 4 methods:
1. Gamma rays: most common method; requires radioactive source
2. Electron beams: lower penetration power
3. X rays: slower than electron-beam irradiation
4. UV light: low penetration, only suitable for surface cleaning
79
Radiation Sterilisation - 7 facts
1. Main target is DNA - but membrane damage may also contribute
2. Direct damage through ionisation, or indirect through radiolysis of water
3. Resistance decreases with moisture or dissolved oxygen
4. Most vegetative cells are sensitive to radiation
5. Bacterial spores & viruses most resistant
6. Dose chosen on safety margin, no consideration for product stability, possible damage, but BP requires no product degradation
7. Used for eg:
- Disposable plastics
- Decontaminate raw materials
- Radiation-stable ointments, some solutions e.g. eye drops
80
Filtration - 7 facts
1. Sterilisation: removal of bacteria, fungi etc
2. Clarification: removal of possibly hazardous particulates
3. Only for liquids or gases/air
4. Cold process: good for heat sensitive products (e.g. biopharmaceuticals)
5. Fast
6. Aseptic process: requires special operation and control
7. Not an absolute process: may be breakthrough & grow through
81
Filtration - 5 Mechanisms
1. Adsorption - due to charge
2. Sieving - exclusion due to dimension of particles (screen filter): absolute filtration rating but low holding capacity (clogs up easily)
3. Trapping in filter matrix (depth filter): no absolute rating but higher capacity
4. Dead-end filtration: fast, but “filter cake” builds up - requires cleaning or replacement
5. Crossflow filtration: slower, but self-cleaning
82
Filtration - 4 materials
1. Polyether sulfone (PES):
Wide pH range, low protein adsorption
2. Cellulose acetate (CA):
Thermal stability, low adsorption
3. Nylon:
Resistant to solvents & alkaline solutions
High non-specific binding; could lose important substances
4. Polytetrafluorethylene (PTFE):
Hydrophobic - mainly for gas/air
Useful at high temperatures, resistant to solvents & acids
83
Describe: Filtration breakthrough & how it may be counteracted
Most membrane filters are not true screen filters, but depth filters with well defined filtration rating & a capacity limit.
Ergo, some microbes or particles may pass through a filter (breakthrough), depending on:
- size/[] of contaminant
- volume
- flow rate
May be counteracted with:
- Use raw materials with low bioburden
- use 2 or 3 filters in series
84
Describe: Filtration grow through & how it may be counteracted
- If left in contact with membrane filter for long time, bacteria will divide & grow in & through the filter.
- Practical solution: limit time of filtration process, roughly 4 hours.
85
New technologies:
High intensity light:
- Useable for water or injectables; but liquid & container must be must be UV-transparent
Low-temperature plasma:
- Gas subjected to electrical field
- Generates ionised molecules
- Mainly for medical devices - not suitable for e.g. liquids or powders
86
7 Step Sterilisation process
1. cleaning/decontamination
2. inspection & assembly
3. packaging
4. steriliser loading
5. sterilisation cycle
6. storage & distribution
7. record keeping
87
Describe: Quality control, quality assurance, and its importance
- Quality control involves testing of the final product
- Quality assurance is designed to assure that all stages of the manufacturing process contribute to quality of the end product
- Pharmaceutical sterile products are difficult to test; thus quality assurance
88
Validation of sterilisation processes: 5 step process
Demonstrating that a process will consistently produce the results that it is intended to do:
1. calibration equipment
2. testing quality of steam, leak tests
3. software testing
4.physical, chemical & biological indicators
5. comprehensive documentation
89
Validation & control: The 3 areas controlled
1. Environmental control
2. Control of the sterilisation process
3. Control of the final product
90
Environmental control
air:
- settle plates (contamination of air)
- air samplers
surfaces:
- contact plates
- swabbing
91
Control of sterilisation processes' 3 methods
Methods:
1. indicators (physical, chemical, biological)
2. parametric release
3. sterility tests
92
4 Physical indicators
1. Heat sterilisation - digital record of temp, taken at coolest part of loaded steriliser
2. Gaseous sterilisation - leak tests, pressure testing
3. Radiation - plastic dosimeter darkens in proportion to radiation
4. Filtration - bubble point pressure test
93
3 Chemical indicators
1. Usually undergo melting or change of colour that can be observed
2. Not necessarily corresponds to microbiological sterility
3. Use of autoclave tape, or Browne's tubes
94
Describe: Browne's tubes
Browne’s tubes:
- Upon heating an acid is produced
- Indicator changes colour
- Speed of reaction determined by temperature
- Different types for moist/dry heat sterilisation
95
3 Biological indicators
1. Biological indicators are only included when validating a method, not as a routine control indicator
2. Suspension of bacteria in water/media OR spores dried on strip of e.g. paper
3. After sterilisation incubated: if turbid, sterilisation failed
96
Sterility test
- Limited number of items tested for presence of bacteria
- Only recognises organisms that grow under conditions of test
- Sample size is restricted
- Sampling itself may introduce contamination
- Must ensure all samples in batch treated similarly
- Passing the sterility test does not mean the batch is sterile
- Sterility test will only detect gross failure
- Used as additional check, not on its own
97
Sampling
p - probability contaminated
q - probability not contaminated
p + q = 1 and q = 1 - p
if p = 0.1 (10% contaminated); q = 0.90
2 samples, both non-infected: --> q2 = 0.9*0.9 = 0.81
n samples, all non-infected --> qn
98
Parametric release and the validation studies it requires
- A system of release assures product is intended quality, based on data collected during manufacturing instead of sterility testing.
- All relevant parameters (temp, pressure, time etc) must be accurately controlled & measured
- Parametric release is only applied to products that are terminally sterilised in their final container
Requires validation studies such as:
-heat distribution and penetration
- bioburden
- container closure
- cycle lethality studies
- Requires pre-sterilisation bioburden testing of each batch
- Each cycle includes chemical or biological indicators
- Authorisation requires historical batch data
99
Pyrogen removal and the 3 testing methods
High Heat
Distillation
Ultrafiltration
Ion-exchange chromatography
Alkali or oxidising agent treatment
1. Rabbit pyrogen test (RPT)
2. Limulus amoebocyte lysate (LAL) test
3. Monocyte Activation Tests (MAT)
100
Rabbit pyrogen test & drawbacks
3 animals injected, then the temp recorded over 3 hours
Success or fail dependant on temp changes, may use additional rabbits based on failure
Drawbacks:
- not very sensitive
- may depend on age/gender/species of rabbit
- repeated use of animal leads to endotoxin tolerance
- low reactivity to endotoxin from certain bacteria (e.g. Legionella)
- some drugs may influence body temp.
- not quantitative (only pass/fail)
101
Limulus amoebocyte lysate test & drawbacks
- blood cells isolated from horseshoe crabs (Limulus polyphemus)
lysate purified - contains enzymes that are activated by endotoxins
- if mixed with endotoxins, coagulation occurs (or in modified tests, a colour change) is observed
Drawbacks:
- detects only LPS
- some compounds may interfere with clotting system
- can only be used if product has pH 6-8
- Alternative now possible: protein from horseshoe crabs (Limulus clotting factor C) that is produced by recombinant DNA technology
102
Monocyte activation test (MAT)
- based on predicting human response to pyrogens
- monocytes (from blood or cell line) mixed with sample
- Cytokine production tested with immunoassay (ELISA)
103
Sterility Assurance - 4 facts
1. Expressed as Sterility Assurance Level (SAL) or Probability of a Non-Sterile Unit (PNSU)
2. The probabilities a microorganisms may survive in an item, batch, or unit, a microorganism survives
3. A SAL of 10-6 is considered acceptable
4. Sterilisation procedures based on “worst case” scenario: sterility assurance level achieved is better than required
103
Sterility Assurance Level (SAL) - Number
- Important is how many bacteria are in the product before sterilisation (=bioburden)
- e.g. if bioburden = 100 bacteria & time to reduce living bacteria 10-fold is 3 min (i.e. D=3 min)
- 0.1 bacterium = probability of batch contamination is 1/10, meaning the sterility assurance level (SAL) is 10^-1
- e.g.: if initial bioburden is 10^2, & we need a SAL of 10^-6 is needed, then 8 log cycles are required to kill.
1 Log cycle of kill = D value: to get SAL 10^-6, we need 8 x the D value
104
Sterility Assurance level - Calculations
IF=N0/N
10^2/10^-6=10^8
logIF=logN0-logN
logIF=2-(-6)=8
8 log cycles to kill, 8 times D value
t=logIF*D IF=10^t/D
105
Probability of a Non-sterile unit (PNSU)
PNSU: probability that 1 unit in a batch is contaminated.
e.g.: initial bioburden of 102/ml, filled in 1 ml ampoules
PNSU 1 in 10 (=10^-1)
reducing probability to 0.1: IF = 10^2/10^-1 = 10^3 = 3 log cycles of kill
PNSU 1 in 10^6 (=10^-6)
reducing probability to 10^-6: IF = 10^2/10^-6 = 10^8 = 8 log cycles of kill
106
Autoclaving
- Autoclaving time (holding time in the autoclave) may be minimised to acceptable & practical level
- reduce costs
- reduce degradation of drug
107
Contribution of heating-up/cooling down process in autoclaving - 6 facts
1. Heating & cooling stages contribute to the sterilisation process
2. If contribution is known, holding stage can be reduced
3. May save time, costs &, if product is thermolabile, reduce inactivation
4. Convert temp-time combinations to equivalent time at standard temp (e.g.121oC)
5. Can be used to calculate contribution of heating & cooling stages
6. Also used to compare & validate autoclaving cycles at non-standard temp
108
The F-concept - 4 facts
1. F-value: expresses heat treatment lethality at temp equivalent to treatment at a reference temp
2. F-value depends on resistance of reference organism
3. F-value is expressed in minutes
4. Mainly used with moist heat sterilisation, & only if temp is the sole factor influencing the process
109
Lethal rate calculations
Lethal rate (L) = Ftref/FT = 10*(T-Tref/z)
FT=DT*(logN0-logN)=DT*logIF
For instance, sterilising at:
111C : L = 0.1 (i.e. sterilise 10x longer for same lethality)
131C: L = 10 (i.e. sterilise 10x shorter for same lethality)
Example: Calculate autoclaving time at 112C to give an F0 of 12 min
L=F0/FT, so L=10^(112-121)=0.126
F0/L=95min
110
List 4 things desired form an excipient
1. Cheap / inexpensive (mass market pharma)
2. Biologically inert – no physiological effects
3. Chemically inert – no reaction with API
4. Able to produce range of physico-chemical properties – e.g. viscosity and/or solubility
111
As an excipient: Lactose - 4 facts
1. Several different forms (chemical, physical particle size, degrees of hydration etc.)
2. Extremely prevalent in pharmaceuticals (tablets)
3. Mainly a filler and/or binder. Naturally sweet.
4. Glucose can be either a or b (galactose always b)
112
Polysaccharides as excipients - 4 facts
1. Modified cellulose derivatives, gives a range of physico-chemical properties
2. Cellulose: ethers, esters, oxidised cellulose (changing cellulose chemistry = change chemical properties).
3. Conformational constraints & intramolecular H-bonding
4. Intermolecular (molecule-molecule) H-bonding to other cellulose molecules gives rise to quasi-crystalline microfibrils
113
Microcrystalline Cellulose (popular excipient) - 5 properties
1. Wide range of particle sizes
2. Very low coefficient of friction
3. High compressibility to give a strong compact
4. Compact disintegrates freely
5. Diluent, disintegrant & lubricant
114
The methyl ether – Methylcellulose - 4 facts
1. Swells in cold water to give a clear, viscous solution, useful as a thickener.
2. If taken in relatively large doses, bulks up in the gut, therefore a ‘bulk-forming laxative.'
3. Insoluble in hot water & most organic solvents
4. Dispersant, thickener & emulsifier in many liquid preparations
115
Hydroxyethyl cellulose - 5 properties & 3 uses
1. Soluble in water to give a clear viscous solution, thickener
2. Non-ionic
3. Soluble in water <40C, insoluble >45C
4. Soluble in polar organic solvents
5. Tableting binder & granulating agent
1. Alcohol-soluble thickener & suspending agent in liquid preparations
2. Tablet binding
3. Ophthalmic solutions
116
Starch as an excipient - 2 uses
1. Filler, glidant, disintegrant in tablets
2. Suspending agent
117
Alginates as excipients - 4 uses
1. Sodium alginate is used as a stabiliser in emulsions etc
2. Sodium alginate is used in some commercial antacid preparations
3. It's a ‘raft-forming’ treatment for reflux etc.
4. Calcium alginate can be formed as fibres & woven into a fabric for use as an absorbable haemostatic dressing
118
Define: Disinfection
Disinfection: Process of removing micro-organisms from surfaces of inanimate objects. Not the same as sterilization.
119
How are disinfectants categorized, and what are the three categories?
Different types of disinfectants are categorized according to their capability:
1. High level
2. Intermediate level
3. Low level
120
High level disinfectant
High level (Chemical sterilants): e.g. Aldehydes, bleach
Destroys: ALL micro-organisms
Excludes: prions (misfolded proteins)
121
Intermediate level disinfectant
Intermediate-level: e.g. Alcohols, Biguanides,
Destroys: ALL bacteria, Most viruses & fungi
Excludes: some viruses, bacterial spores, prions
122
Low level disinfectant
Low-level: e.g. Phenolics
Destroys: Most bacteria, Some viruses, fungi
Excludes: TB, some viruses and fungi, bacterial spores, Prions
123
6 Ideal properties of disinfectants:
1. Broad spectrum
2. Rapid kill (5 mins)
3. Must not damage material to be disinfected
4. Easy to prepare & use
5. Stable
6. Inexpensive
124
Antisepsis
Antisepsis: The destruction or inhibition of micro-organisms on living tissues.
- Community setting: Decrease incidence of wound infections
- Hospital setting: Decrease HAI
- decrease normal skin flora prior to surgery
1. pre-op surgical scrubs
2. patient pre-operation skin preps
- Decrease of infection by hand washing
125
Resident bacteria, Transient bacteria and Antiseptics aim
Antiseptic relation with Bacteria on our skin:
Resident bacteria - Gram positive e.g. Staphylococcus aureus,
Transient bacteria - Gram negative e.g. E. coli
Antiseptics aim to remove the transient & resident micro-orgs on our skin- Need a broad spectrum of activity
126
7 Ideal properties of antiseptics
1. Adequate spectrum anti-microbial activity
2. Immediate activity - active within 1 min
3. Persistent activity – how long activity is retained
4. Cumulative (residual) activity – antimicrobial effects when used repeatedly
5. Non-toxic, non-irritant
6. Cost
7. Motivation to use
127
2 Common antiseptics: immediate, persistent, residual
Activities of Common Antiseptics (Immediate, Persistence, Residual)
4% chlorhexidine gluconate (CHG)
Immediate: High Persistent: High Residual: High
95% Alcohol & 0.5% chlorhexidine glucoate
Immediate: High Persistent: High Residual: High
128
6 Factors affecting choice of chemical agent ‘(Antiseptics and Disinfectants)’
1. Intended application
2. Number of micro-orgs present
3. Type micro-orgs present
4. Environmental factors
5. Safety, stability, cost, ease of use
6. Properties of chemical agent
129
3 Factors affecting choice of chemical agent: Intended application
Disinfection:
1. Degree of killing required
- sterilisation vs. high level or medium/low level disinfection
- Cleaning is as important as disinfection
2. Composition of surface to be disinfected:
- degradation following prolonged regular use
- non-specific absorption by rubber & plastics
- accessibility to surface (hard to reach?)
3. Antisepsis: Toxicity: Risk increases if chemical applied to broken skin
130
3 Factors affecting choice of chemical agent: Number of micro-organisms present
1. large number of micro-organisms = High bioburden
2. High bioburden requires:
- [higher]
- longer exposure times
3. Biofilms are 100x more resistant to disinfectants.
131
Hardest to Easiest type of micro-organism to remove
Hardest to remove:
Prions (Creutzfeldt-Jacob Disease, BSE)-Protein
Spores (Bacillus sp. C. difficile)
Mycobacteria
Small non-enveloped viruses (Polio)
Gram-negative bacteria
Fungi (Candida)
Large non-enveloped viruses (Enterovirus)
Gram-positive bacteria (incl. MRSA, VRE)
Lipid enveloped viruses (HIV, HBV)
Easiest to remove
132
2 Factors affecting choice of chemical agent: Environmental factors
Organic matter (blood, pus, food residues, dirt) can decrease antimicrobial capacity:
1. Chemically inactivates disinfectant
2. Absorption to the bacteria surface, masking bacteria
e.g. Cl2 & I2 based compounds are very susceptible (e.g. Bleach).
Divalent cations (Ca2+, Mg 2+) - decrease activity:
1. stabilise bacterial cell wall
2. block disinfectants adsorption sites
( can use chelating agents to ‘mop up’ cations and increase efficacy)
133
5 Factors affecting disinfectant choice: safety, stability, cost, ease of use:
1. Cost effective
2. Motivation to use
3. Some are toxic & volatile, requiring extractor facility & skin protection/ Masks e.g. aldehydes
4. Antiseptics: more risk when applied to broken skin/mucous membranes
5. Stability (some compounds are unstable):
- Unstable compounds stored as INACTIVE form
- Activate prior to use (eg Bleach needs water to activate)
134
Factors affecting choice of disinfectants: properties
1. Concentration / Dilution
2. Temperature
3. pH
4. Exposure time
135
Dilution - 3 facts
Dilution:
1. A higher [disinfectant] increases efficacy & decreases exposure time required to kill micro-organisms.
2. But dilution doesn't affect killing capacity of all disinfectants equally.
3. Some chemical agents are more effected by dilution than others.
136
Dilution coefficient
Dilution coefficient: a number that expresses the effectiveness of a disinfectant for a given organism. The gradient of the line of the log D value against the [log].
h = Log D value at conc. 2 – Log D value at conc. 1 all divided by
(Log conc. 1 – Log conc. 2)
The higher the Dilution coefficient the more effected the chemical is by dilution
h =1 1 in 2 dilution = 21 decrease in activity of antimicrobial
h = 6 1 in 2 dilution = 26 (64x) decrease in activity of antimicrobial
137
Antiseptics - pH: 2 facts
pH:
- Affects survival and growth of organism
Affects potency; dependent on chemical, due to:
1. ionisation
2. polymerisation
3. binding to cell surface
138
Antiseptics - Exposure time: 2 facts
Exposure time:
- Longer exposure times increases efficacy
- Antimicrobial must remain wet, consider drying time [&]
139
Suspensions test
Suspension test:
Dilutions of disinfectant added to standardised bacterial suspension (number and type) + albumin (to mimic dirty environment).
Incubate at a set temp.
At a specific time interval:
- remove sample,
- neutralise disinfectant
- determine viable count (cfu/ml)
Calculate conc. disinfectant req. to kill 99.999%
(5 log kill; 1 in 100 000 bacteria survive)
140
Suspension test: 4 Validation requirements
FOR THE TEST TO BE VALID
- Know viable count of inoculum
- Experimental conditions validation
- Neutraliser toxicity validation
- Disinfectant neutralisation validation
The suspension test is a quantitative test.
It will test the efficacy of disinfectants and antiseptics
It will give us a concentration for each type of bacteria tested.
141
Test - Minimum Inhibitory Concentration (MIC):
For each micro-organism to be tested:
Prepare doubling dilutions of test agent in culture medium
i.e. 1/2; 1/4; 1/8; 1/16; 1/32; 1/64, 1/128
Inoculate with test organism.
Incubate at 37C overnight
Score for growth
MIC is lowest concentration showing no growth
142
Qualitative test - Disc test
Agar spread with test organism, incubate at 37C overnight
Filter paper disc impregnated with known conc. test agent placed on plate
Zone of growth inhibition indicates antimicrobial activity. Measure. Compare to standards.
143
Evaluation of Antiseptics: Time Kill:
Allows evaluation of antiseptics
Time intervals are short
Ideal kill time for an antiseptic is less than 1 minute
Repeat regularly – monitor changes in time to kill- development of resistance?
Time Kill:
Quantitative method particularly useful for antiseptics
144
Define: Contact time
Contact time – how long a disinfectant must stay wet on a surface to do its job
145
Aliphatic alcohols
Aliphatic Alcohols
e.g. Ethanol, Isopropanol
Uses as 50-90% soln, 100% can’t permeate bacteria
Antiseptic- skin cleansing
Intermediate disinfection (clean surfaces)
E.g. Aseptic manufacture
146
Aliphatic alcohols: 5 Adv vs 6 Dis
Adv:
1. Broad antimicrobial activity - active against bacteria (incl. mycobacteria) & fungi
2. Rapid kill
3. Water soluble
4. Relatively non toxic
5. No residues
Dis:
1. Not sporicidal
2. Isopropanol not viracidal
3. Poor penetration of organic matter
4. High dilution coefficient (used at >50%)
5. Flammable
6. Expensive
147
Aldehydes: Disinfectants
Aldehydes: e.g. Glutaraldehyde* (2%) , (liquid)
Formaldehyde (2-4%), (gas or liquid)
Ortho-phthalaldehyde (OPA; 0.5%) liquid
Not used as an antiseptic. Uses – sterilisation & high level disinfection
148
Aliphatic alcohols: 4 Adv vs 5 Dis
Adv:
1. Broad spectrum
2. Sporicidal
3. Rapid kill
4. Not affected by organic matter- use in dirty areas
Dis:
1. Unstable:
2. Activity decreased by polymerization
3. Supplied as 2% soln in acid
4.Activated with alkalising agent
5. Toxic
149
Biguanides - Disinfectant
Biguanides e.g. Chlorhexidine (Chlorhexidine Gluconate)
Uses: antiseptic, preservative e.g. mouth wash
150
Biguanides: 2 Adv vs 5 Dis
Adv:
1. Good bactericidal activity
2. Nontoxic
Dis:
1. Not active against mycobacteria
2. Not sporicidal or viracidal
3. Not v. soluble in water
4. Activity decreased by anionic compounds eg soaps
5. & decreased by organic matter
151
Chlorine Halogens: Liquid Chlorine: 3 Adv vs 1 Dis and its uses
Chlorine Halogens: Liquid Chlorine
Adv:
1. Broad spectrum
2. Active against bacteria, fungi, viruses, spores
3. Rapid Kill
Dis:
1. Irritant
Uses: Water purification, High level disinfection
152
Chlorine Halogens: 4 Adv vs 4 Dis
Chlorine Halogens: Hypochlorite (bleach) HOCl reversible OCl- + H+
Uses: Disinfectant* 1-10% solution for surfaces / instruments
Antiseptic: 0.5% solution for skin and wounds
Adv:
1. Readily available
2. Cheap, Compatible
3. Broad spectrum - Active against bacteria fungi, viruses (& spores)
4. Rapid kill
Dis:
1. Corrosive
2. Irritant
3. Inactivated by organic matter
4. Unstable (make it up as you want to use it)
153
Iodine halogens: Uses, 3 Adv vs 2 Dis
Iodine Halogens:
Uses: Disinfectants and Antiseptics* eg prior to surgery
Advantages:
1. Broad spectrum (bacteria, viruses, fungi (& spores))
2. Rapid kill
3. Less affected by pH, temp & organic matter than Cl2 based compounds
Dis:
1. stains skin & fabric
2. irritant
154
Iodophores: 5 uses
Iodophores: e.g. povidone-iodine (10% soln); betadine
Allow slow release of I2 from a complex
Limits staining but requires a longer contact time
Uses:
1. Topical application
2. Surgical scrub
3. Pre & post operative skin cleansing
4. Safe on mucous membranes
5. Impregnated into wound dressings
155
Heavy metal antiseptics: Uses
Heavy metals – Silver
Broad spectrum antimicrobial
Non-toxic BUT expensive
Uses:
Antiseptic e.g. Silver nitrate, Silver sulfadiazine
Impregnated into wound dressings
- management of acute and chronic wounds & burns- (inhibition of wound healing?)
Impregnated into indwelling medical devices
- urinary catheters decrease CA-UTI
- intratracheal breathing tubes decrease VAP
- Bone protheses decrease infection after joint replacement
156
Peroxygen compounds: 4 Adv vs 3 Dis
Peroxygen compounds: e.g. hydrogen peroxide:
high [Sterilant], low [disinfectant, antiseptic]
Sterilant [0.1-0.35%]
Adv:
1. Powerful antimicrobials & sporicidal
2. Active in presence of organic matter
3. Break down to non-toxic products
4. No residue
Dis:
1. Unstable in sunlight
2. Irritant
3. Corrosive
157
Phenolics: 2 Adv vs 5 Dis
Phenolics:
Adv:
1. Good antimicrobial activity; poor sporocide
2. Cheap
Disadvantages
1. Activity decreased by dilution
2. Activity of some products decreased by organic matter / increasing pH
3. Some harmful / toxic
4. Some have strong odour (ever smelt TCP?)
5. Some corrosive
158
Substituted phenols: Phenolic tar acids: 4 Adv vs 2 Dis
Substituted phenols: Phenolic tar acids (coal tar derivatives); Ethyl phenols - hospital disinfectant
Adv:
1. Broad spectrum
2. Rapid kill
3. Cheap
4. Not affected by organic matter
Dis:
1. NOT sporicidal
2. Leave residues
159
Substituted phenols; Non-coal tar halogenated phenols: 2 Adv and use
Substituted phenols; Non-coal tar halogenated phenols:
Adv:
1. Broad spectrum, stable
2. Non-toxic, active at [low]
Uses: antiseptics & preservatives
Triclosan is an anti-bacterial agent used in cleaning and a preservative, but resistance is building against it
160
Cationic surface-active agents: QACs (Quaternary ammonium compounds): Use and 6 Adv vs 3 Dis
Cationic surface-active agents: QACs (Quaternary ammonium compounds)
Uses: disinfectants, antiseptics, preservatives
Adv:
1. Water soluble.
2. Stable,
3. Non-toxic,
4. Non-corrosive
5. Broad spectrum
6. Active at low conc.
Dis:
1. Not sporicidal.
2. Activity decreased by organic matter
3. Can leave a residue
161
Disinfectants 3 Requirements for antimicrobial activity:
Requirements for antimicrobial activity:
1. Adsorption to cell surface- (requires moisture- must be wet)
2. Passage into cell- (structure of outer surface effects sensitivity to disinfectants)
3. Interaction with target- (disinfectants have a specific target (eg ribosme/ nucleoid etc)
162
What are the four modes of action for disinfectants?
Mode of action:
1. Oxidation
2. Crosslinking
3. Coagulation
4. Disruption of structures
Some disinfectants may have multiple modes of action.
The Mode of Action is not clearly defined for all our disinfectants.
163
Disinfectant mode of action: Oxidation's 3 effects
Oxidation leads to disruption & breaking of bonds in macromolecules.
1. Some chemicals bind tightly to DNA/RNA causing strand breakage, disrupting processes such as translation& transcription, & prevents cell division.
2. Unsaturated fatty acid degradation in membrane disrupts permeability, causes leaks leading to cell death.
3. Disulphide bond modification in proteins & enzymes disrupts function & kills cells.
164
Disinfectant mode of action: X Linking's 2 effects
X-linking
- Aldehydes cause X linking of lysine residues to other amino acids- changes protein structure & causes protein aggregation.
- Cross linking of DNA, RNA/ proteins/ peptidoglycan
165
Disinfectant mode of action: Coagulation's 1 effect
Coagulation
- Macromolecule denaturation– leads to extensive coagulation & precipitation, (disruption of cytoplasm, lipid membranes etc)
166
Disinfectant mode of action: Disruption of functional structures ( cell wall/ cytoplasmic membrane) - 2 versions
1. + Ions have affinity for - microbial membranes
This affinity causes disruption of the proton motive force
Disrupts cell membrane-associated activities e.g. energy generation
Proton motive force: Electrochemical gradient across membrane allowing proton movement across. Cell can’t function without it.
2. Heavy metals:
Directly bind to & damage lipid membrane
Indirectly bind to LPS on Gram + bacteria
167
4 Mechanisms of resistance
1. Intrinsic/ innate resistance: a natural chromosomally encoded property
2. Extrinsic/ acquired resistance: organisms become resistant
3. Phenotypic: response to mode of growth (reversible)
4. Genetic: mutation (irreversible) ,genetic transfer (e.g. plasmid)
Includes co-resistance (multiple resistance genes)
168
Efflux pumps
1. Efflux pumps remove antimicrobials from cell.
2. Agent doesn’t reach [optimum]
3. Gives resist to unrelated compounds
169
2 forms of Phenotypic resistance
1. Parasite within protozoa are more resistant then planktonic bacteria
2. Biofilm develops, providing barrier
Facilitates cell-cell communication
Community develops, Increasing genetic exchange
170
Acquired genetic resistance: chromosomal mutation / gene transfer: The 5 changes which can decrease efficacy of antimicrobial agent:
Changes which can decrease efficacy of antimicrobial agent:
1. protein and phospholipid composition
2. amount & size of porins
3. modified target
4. modified metabolic pathway
5. Increase efflux
171
Acquired genetic resistance via efflux:
e.g. upregulation AcrAB-TolC system in E. coli
Widespread resist to multiple antimicrobials
Partly controlled by MAR (multiple antibiotic resistant) operon
2 components change pump expression:
mar A – activator
Mar R – repressor
Inactivation of R or over expression of A increase pump efficiency
Change caused by mutation
