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Sterilization and validation

Sterilization and validation. Richard Marchand MD Medical Microbiologist and Infectious Diseases Assistant Professor University of Montreal One hospital (The CSSSSVLDLJ) Somewhere in Quebec. Plan. History Basic definitions A lot of little quizzzz Properties of heat, steam and bugs

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Sterilization and validation

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  1. Sterilization and validation Richard Marchand MD Medical Microbiologist and Infectious Diseases Assistant Professor University of Montreal One hospital (The CSSSSVLDLJ) Somewhere in Quebec

  2. Plan • History • Basic definitions • A lot of little quizzzz • Properties of heat, steam and bugs • Process validation • Biological Indicators • Chemical Indicators • Water, water, water…

  3. Disinfection or Sterilisation ? • What is the needed temperature to inactivate bacteria ???? Answer : It depend on the bacteria because some are highly resistant to heat. (Ex.: Geothermal bacteria like hot spring bugs withstand 250 oC ) Most human pathogens were thought to be killed by temperature lower than 150 oC.

  4. Little Quizz • Question : Where came from these weird standards like • Reduction of 106 • Temperatures of 121, 132 and 134 oC ????? • Answer: From the post world war II food industry (mostly « canning ») FDA : FOOD and Drug Agency

  5. Steam sterilization origins

  6. HISTORY • 1862 Invention of the Autoclave • 1880 The first indicator : a potato • 1906 Creation of the FDA • 1925 Waxy pellets that melt at 121oC • 1932 (ATI) CI with lead sulphite passes from black to white • 1940 (ATI) CI Chromium TriChloride passes from purple to green

  7. Pasteurisation and tyndallisation • Around 1860 Louis Pasteur demonstrated that the heating between 50 and 60 oC without air for 30 minutes prevent deterioration of wine during transport. He also demonstrated that a previous heating of the malt before yeast inoculation prevented beer contamination. • Later a process called tyndallisation was developed by Tyndall and consisted in a series of burst of increased temperature up to 70 oC at regular intervals (originally once a day for 3 days). This is to activate the resistant forms to germinate in order to kill them with the next heat burst. Milk pasteurisation was to follow by using 30 minutes heat burst at 63 oC followed later by a 15 minutes heat burst at 73 oC.

  8. HISTORY • 39-45 World War II (many cases of food poisoning) • 1950-60 Development of validation concepts for the canning industry (Clost. bot) • 1960-70 Development of the Dvalue conceptusing heat resistant spores

  9. The canning industry monitoring • Spores (Bacillus and Clostridia) are the most heat resistant organisms • Spores are to be killed with a good and reliable probability So • Lets use non infectious spores to ascertain that the beans and the sardines are safely canned

  10. How does heat kill micro organisms ? • Heat coagulates proteins : egg white • Heat has a mild oxidative effect What is oxidation ? Will discuss that later

  11. Water Boils at 100 °C : fresh water in a pan with a lid 97 °C : fresh water in a pan without a lid 104 °C : sea water (depending on salt concentration) • Boiling never guarantees that the temperature was high enough for bacterial proteins to coagulate • The very low water content of spores and other substances (« Heat shock proteins » also called “stress proteins) protects them from denaturation • Sporulated pathogens (like Clostridium sp.) resists up to 8H30 at 100°C. (Lowering risks of infections other than tetanus or gas gangrene )

  12. Boiling Temperature is linked to pressure • 100 oC at 1 ATM • 121 oC at 2 ATM (1 barr) • 132 oC at 3 ATM (2 barr) • 80 oC at 0.5 ATM (Mont Blanc) • 70 oC at 0.35 ATM (Mont Everest) • 40 oC at 0.02 ATM (Mechanical vacuum)

  13. Quizz Pasteur ? Why 2 or more exposures to heat ? Answer : For germination to happen Why « no air » ? Answer : Because the air blocks the energy transfer toward organic matter.

  14. The origin of Biological indicators BI • Although most do not, some bacterial species die in a predictable manner, specially some sporulated thermophillic bacilli So lets screen them and use the most resistant and predictable safe bug that can be “canned” to design safe processes.

  15. SURVIVAL PROBABILITY

  16. How to define resistance to heat ? • The D value concept was invented !

  17. D value: principle and logic • More it is hot, faster the micro organisms die • Faster the bugs die, faster is the process • The speed of the process is expressed with the D value • D value = exposition time required (in minutes) to kill 1 log (90%) of the micro organisms • A Dvalue of 6 means that it takes 6 minutes to reduce par a factor of 10 (90%) or 1 log the number of micro organisms. N.B. Faster does not mean more efficient, because a bug killed more slowly is not less dead.

  18. Effect of probabilityfor a BI exposed to heat For a BI of : 2.0 X 106 et Dvalue* of 1 minute • Survival • After 1.0 minute = 200,000 • After 2.0 minutes = 20,000 • After 3.0 minutes = 2000 • After 4.0 minutes = 200 • After 5.0 minutes = 20 • After 6.0 minutes = 2 • After 7.0 minutes = 0.2 • Total time to reduce to zero = 6.5 minutes *At that time the most resistant Bacillus know was the stearothermophilus with a Dvalue of 1 to 1.5

  19. Effect of probability for many cans Cycle of 6.5 minutes • 1 can/1 BI (2.0 X 106) = 0-0.2 survivor • 10 cans/10 BI (2.0 X 107) = 2 survivors (or á to 7.5 min) • 100 cans/100 BI (2.0 X 108) = 20 surv. (or á to 8.5 min) • 103 cans/103 BI (2.0 X 109) = 200 surv. (or á to 9.5 min) • 104 cans/104 BI (2.0 X 1010)= 2000 surv. (or á 10.5 min) • 105 canss/105 BI (2.0 X 1011)= 20000 surv.(or á 11.5 min) More we have cans, more we have contaminated cans !

  20. Then what about safety ? • Cans batches are generally less than a million at a time So lets double the time, to bring back the survival probability once again around 0 – 0.2 (The overkill approach was born)

  21. Why most micro organisms do not die in a linear fashion ? • The mechanisms of death are not mono molecular (different proteins are degraded or coagulated at different speeds) • The proportion of life essential proteineous “targets” varies with the age of the micro organisms, their life cycle, their food supply etc.. • Susceptibility to heat varies thereof none linearly therefore with limited predictability

  22. Why is it written everywhere that all bugs die on linear scale fashion ? • The fifty percent principle applies • (eg. Half of what is written in textbooks is false, the problem is we don’t know which one) Confusius • Evidence base medicine is applied • (eg.: A concept is an evidence when everybody say the same thing, true or not.) • In fact most European textbooks recognize this fact, while only few American books does

  23. Pasteurisation yesterday • In 1964 it was demonstrated that hotter but shorter heat bursts have less deleterious effect on organic material without loosing its effects on microbial flora. • HOWEVER : This process is not a sterilization process because it kills only the heat sensitive flora.

  24. HISTORY cont. • 1960-70 Development of the Dvalue conceptusing heat resistant spores for sterilisation + Fvalue + Zvalue • 1965 Proposal by Sweden of the SAL for a definition of sterility • 1979 Proposal by Canada of a legal definition of sterility

  25. Z value: principle and logic • If the temperature is lowered, bugs are killed more slowly and the D value increases (because it takes more time to kill) • Conversely if the temperature is higher, faster the bugs die, and lower is the D value • Z value = the number of degree of temperature required to obtain a variation of 1 log of the D value • For a given micro organism, A Zvalue is a measure of its resistance to heat because higher the Zvalue, more heat is needed to augment the Dvalue by a factor of 1.

  26. F value: principle and logic • If the D value is measured at different temperature and pressure it can be seen that a D value varies with the pressure. More the Dvalue decreases with a specific increase of pressure, more powerful is considered the process. • F value = a measure of the capacity to inactivate bacteria in function of the temperature. • Mathematically the F value is expressed by the rate of mortality per minute in function of temperature for a given pressure. • This concept applies de facto to steam sterilization only and is a measure of the power of a sterilizer (Big boilers and big pipes are faster than a kettle.)

  27. Little quizzz Question : Is all processes D values a time dependant measure ? Answer : No, for radiation and ozone sterilization the Dvalue is dose dependant

  28. The reference cycle 1965 The Swedish National Health Board proposed : Sterility Assurance Level (SAL) 10-6 • 121 oC (gravity) and 1.05 bar • 106 spores with a Dvalue of 1.0 to 1.5 min • Overkill Cycle of 12 to 18 minutes + conditioning and rising time (Temperature and Pressure ) • At that time because of gravity cycle technology, average cycle took : 30 minutes

  29. SURVIVAL PROBABILITY

  30. STERILITY : LEGALDEFINITION proposed by Canada • A medical device can be qualified of sterile if: the probability of survival of a micro organism is less then : • «FOR IMPLANTABLES» : 1 on 1,000,000 (SAL of 10-6) • «FOR TOPICALS» : 1 on 1000 (SAL of 10-3) • + 2 other conditions : endotoxins and biomechanical properties SAL = Sterility Assurance Level

  31. STERILITY ASSURANCE LEVEL

  32. Effect of probabilityfor a BI For a BI of : 2.0 X 106 et Dvalue of 1 minute • Survival • After 1.0 minute = 200,000 • After 2.0 minutes = 20,000 • After 3.0 minutes = 2000 • After 4.0 minutes = 200 • After 5.0 minutes = 20 • After 6.0 minutes = 2 • After 7.0 minutes = 0.2 • Total time to reduce to zero = 6.5 minutes

  33. Effect of probability for many packs Cycle of 6.5 minutes • 1 pack/1 BI (2.0 X 106) = 0-0.2survivor • 10 packs/10 BI (2.0 X 107) = 2survivors (or á to 7.5 min) • 100 packs/100 BI (2.0 X 108) = 20 surv. (or á to 8.5 min) • 103 packs/103 BI (2.0 X 109) = 200 surv. (or á to 9.5 min) • 104 packs/104 BI (2.0 X 1010)= 2000 surv. (or á 10.5 min) • 105 packs/105 BI (2.0 X 1011)= 20000 surv.(or á 11.5 min) More we have packs, more wont pass !

  34. What should be done to insure safety with big loads ? • Lets double the time (overkill approach) • BUT : many devices cannot withstand a doubling in time ! • Cannot be applicable by the industry for many things (Industrial sterilizers can handle thousands of BIs) • What if the real bioburden is greater than a million • What if the bugs are dead but their toxins still there ? • The bioburden evaluation approach was born when regulatory bodies agreed to adapt “normalized” and individualised cycles to devices characteristics and lots

  35. The bioburden method in short • Is not application to usual hospital settings • Require microbiological evaluations of the microbial burden on a representative sampling of devices • Require an adaptation of cycles for devices and lot characteristics • Stringent monitoring of all critical parameters

  36. How does the SAL should apply to hospital “overkill” cycles ? • First : do not take into account the conditioning and rise up phases (they add up on the killing therefore increasing the safety margin) • Second, because an overkill approach is used, make sure that the SAL objective is attained before doubling the time • This means that the 10-6 objective should be acquired at half cycle • The whole cycle should be capable to attained a 10-12 • The cycle should be long enough to give some margin for delayed “heating ups” by heat barriers

  37. Effect of liquids or heat barriers

  38. Quizz Question : Can you name a heat barrier material ? Answer : polymers (plastics) like non porous plastic containers, wood, rubber etc..

  39. Other cycles later developed • Vacuum reduces cycle to approximately 20 minutes. • Taking into account the Fvalue : • 132 oC • 2.0 bar • Time drops to 4 minutes Preferred by many for orthopaedic steel and metallic devices

  40. In the real . . . Sterilization 3.5 to 15 min Pre conditioning Dry time 15 to 20 minutes max.

  41. Why 132 oC for metals ? • Less damaging for the passivation layer • Less oxidation (pin point oxidation) on edges (sharpness is kept longer) • Less water deposition and water stains • Less “fatigue” because of shorter exposure to heat • Less micro fractures • Faster

  42. Pasteurisation cont. : Today= energy bursts of all kinds • Physical methods : heat (with or without steam), UVs, ultrasounds, pressure bursts etc.. • Chemical Methods : peroxyde, ozone, cold plasma, glow discharge plasma etc.. • 132oC for 4 minutes is basically the same concept In all cases, for them to work, water is required to a certain level. No water molecules, to little or to much will hamper these process.

  43. Little quizzzzz : Name the critical parameters of steam sterilization • Temperature (121 oC) • Time (duration) • Humidity • Pressure (includes vacuum) • Phases of cycle (manufacturer and Europe)

  44. 134oC SAL of 10-6 (total cycle of 3.5 minutes) BIOLOGICAL KILL (HALF-CYCLE) 0 minute 1 minute 3 minutes 2 minutes S.A.L. 10-6 This is presuming all mechanical aspects of your process are working the way they should and you are getting adequate saturated steam.

  45. The challenge of short cycles • 1 minute in a 3.5 minutes cycle is a variation of 30 percent • 1 minute in a 20 minutes cycle is a variation of 5 percent Shorter cycles • need a much more precise monitoring and highly sensitive indicators • are greatly influenced by heat barriers • are at greater risks of errors if the quality of steam is not optimal • are much more susceptible to condensation if a proper pre-conditioning is not done

  46. A lot of stored energy ! Little Quizzzzzz What is the difference between steam at 100oC and humidity at 100oC ? What is the difference between 0oC ice solid water and et 0oC liquid water ? Can water exist as a gas at temperature lower than 0oC ? Can water not be steam at temperature higher than 100oC ? Yes, humidity

  47. Humidity versus Vapour For a given temperature • Humidity and vapour are both constituted of water molecules in a gas state but, with very different levels of stored energy. All gases in presence of water have some of it in a gaseous state called humidity.

  48. Is humidity a must ? Humidity is an intermediate between the steam (the energy source) and the organic material (the target) Humidity acts as the transient energy buffer transferring state that permits proteins denaturation called “hydrolysis”

  49. Role for humidity • The sterilant (the steam) transport the energy (1kg = 540 kCal) • The energy is not transferred efficiently to the organic matter or the device to sterilise by the air or by a gaz. ( remember dry heat is a poor process) • Lack of humidity = lack of transport buffer = poor energy transfer • Surplus of humidity = difficulty to transfer the energy to the target because you put the energy in the buffer that you have to fill first (the water absorbs the energy of the sterilant)

  50. Dry heat (Purkins 1960) • 170 oC (340 oF) : 60 minutes • 160 oC (320 oF) : 120 minutes • 150 oC (300 oF) : 150 minutes • 140 oC (285 oF) : 180 minutes • 121 oC (250 oF) : 12 to 14 hours

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