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 o C ) Most human pathogens were thought to be killed by temperature lower than 150 o C.

4. Little Quizz Question : Where came from these weird standards like – Reduction of 10 6 – Temperatures of 121, 132 and 134 o C ????? 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 121 o C 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 o C 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 o C 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 o C followed later by a 15 minutes heat burst at 73 o C.

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 D value concept using 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 o C at 1 ATM 121 o C at 2 ATM (1 barr) 132 o C at 3 ATM (2 barr) 80 o C at 0.5 ATM (Mont Blanc) 70 o C at 0.35 ATM (Mont Everest) 40 o C 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 (i n 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 probability for a BI exposed to heat For a BI of : 2.0 X 10 6 et D value* 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 10 6 ) = 0-0.2 survivor 10 cans/10 BI (2.0 X 10 7 ) = 2 survivors (or  to 7.5 min) 100 cans/100 BI (2.0 X 10 8 ) = 20 surv. (or  to 8.5 min) 10 3 cans/10 3 BI (2.0 X 10 9 ) = 200 surv. (or  to 9.5 min) 10 4 cans/10 4 BI (2.0 X 10 10 ) = 2000 surv. (or  10.5 min) 10 5 canss/10 5 BI (2.0 X 10 11 ) = 20000 surv. (or  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 D value concept using heat resistant spores for sterilisation + F value + Z value 1965 Proposal by Sweden of the SAL for a definition of sterility 1979Proposal 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 o C (gravity) and 1.05 bar 10 6 spores with a D value 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 : LEGAL DEFINITION 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 probability for a BI For a BI of : 2.0 X 10 6 et D value 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 10 6 ) = 0-0.2 survivor 10 packs/10 BI (2.0 X 10 7 ) = 2 survivors (or  to 7.5 min) 100 packs/100 BI (2.0 X 10 8 ) = 20 surv. (or  to 8.5 min) 10 3 packs/10 3 BI (2.0 X 10 9 ) = 200 surv. (or  to 9.5 min) 10 4 packs/10 4 BI (2.0 X 10 10 ) = 2000 surv. (or  10.5 min) 10 5 packs/10 5 BI (2.0 X 10 11 ) = 20000 surv. (or  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 o C – 2.0 bar – Time drops to 4 minutes Preferred by many for orthopaedic steel and metallic devices

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

41. Why 132 o C 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.. 132 o C 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 o C) Time (duration) Humidity Pressure (includes vacuum) Phases of cycle (manufacturer and Europe)

44. 134 o C SAL of 10 -6 (total cycle of 3.5 minutes) 1 minute 2 minutes 3 minutes BIOLOGICAL KILL (HALF-CYCLE) 0 minute 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. Little Quizzzzzz What is the difference between steam at 100 o C and humidity at 100 o C ? What is the difference between 0 o C ice solid water and et 0 o C liquid water ? A lot of stored energy ! Can water exist as a gas at temperature lower than 0 o C ? Can water not be steam at temperature higher than 100 o C ? 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 o C (340 o F) : 60 minutes 160 o C (320 o F) : 120 minutes 150 o C (300 o F) : 150 minutes 140 o C (285 o F) : 180 minutes 121 o C (250 o F) : 12 to 14 hours

51. It takes 538 calories to convert one mL of water at 100 o C to steam at 100 o C 100 200 300400 500 600 700 water Low energy vapour High energy vapour STEAMTEMPTEMP

52. Instruments cause a temperature drop. 100 200 300400 500 600 700 One or two degrees of temperature drop here… Releases hundreds of calories of heat here.

53. The energy contained and released is called : HEAT OF CONDENSATION 100 200 300400 500 600 700

54. To condense or not to con dance 100 200 300400 500 600 700 If to much energy is released the steam goes back to liquid water ( full condensation) = the devices will be wet.

55. To dance rather than condense The best : If enough energy is kept in the system, the energy release is up to a level where the water is still in a gaseous state (preventing water deposition) 100 200 300400 500 600 700 A very fine tuning line to control

56. What conditions favours full condensation ? Cold instruments when the steam comes in (improper pre-conditioning) Low energy steam (insufficiently heated boilers, bad insulated pipes, calcium deposits) Poor quality steam with lots of debris and salts (favours total release of the energy) To much humidity (Wet steam)

57. Steam quality is important  Saturated steam  98% steam, 2% water vapor  Dry steam  Superheated  Wet steam  Supersaturated

58. Wet dreams steam Steam coming into the sterilizer – Improper jacket temperature – Inadequate Steam lines Distance Verticality Isolation, corrosion Simultaneous demands from other departments X

59. Wet steam (cont’d) Position in the sterilizer (cold spots) – Bottom front near the door – Condensation from above Stacked instrument sets Steam trap function – Standing water Bathtub ring

60. STERILISATION VALIDATION Sterilisation validation is arbitrarily laid on the construction of an cycle based on the behaviour of biologic indicators hopped to be « predictable »

61. BASIC USAGE OF BIs For -) cycle developement -) cycle validation -) routine monitoring Cycle development = numeration analysis and negative fraction analysis In accordance with the cycle philosophy Exceeding force method (overkill) Bioburden evaluation method (bioburden)

62. Variability of BI The D value varies in function of the support The D value varies in function of the wrapping (and assembly) Viability varies with the culture media Viability with the recovering technique Viability goes down with time

63. WHAT IS AN INDICATOR ? On paper Self -contained Sealed ampulla (spores + broth) Spores suspension Tube witness (pt of fusion) It is a device conceived to verify if the process operated as expected

64. Validation of biological indicators The reality : We do not use the most resistant organisms The predictive behaviour is generally linear only for one process Manufacturers seldom use more “practical” strains (read : “less linear” strains for more economical, inoffensive, self resistance and stability) There is no such thing as a “universal biological indicator” The choice of any particular strain is therefore a manner of arbitrary choice

65. Rapid BIs and EIs Rapid Bis do the same thing as conventional Bis but give answers in 1 to 3 hours Rapid Bis use chemical or fluorometric markers to signify the presence of living organisms Enzymatic Indicators use enzymes mimicking life essential proteins as inactivation targets. Answers are obtained in 20 seconds (speed for $$$$$)

66. Role of Biological Indicators BIs, albeit their weaknesses are still the best tools to develop and validate the construction of a cycle or a process from beginning to end. BI manufacturing is therefore submitted to stringed norms and regulation by governmental agencies.

67. 3M Rapid Indicator

69. Commercial BI Spores from all spore vendors are produced by 3 major suppliers Manufacturers of BI must specify : Type of bacterial population Quantity of spores Storage conditions and expiration date Usage condition (culture media and incubation time) Performance characteristics

71. Sportrol ( 1.4 X 10 5 D value 1.5 ) Survivors – 0 min 140,000 – 1.5 min 14,000 – 3.0 min 1,400 – 4.5 min 140 – 6.0 min 14 – 7.5 min 1.4 – 9.0 min 0.14 Complete kill is achieved in 8.5 minutes or 70 % of the required time for 12 log (with a Dvalue of 1.0, or 47% with a Dvalue of 1.5)

73. Attest ( 2.5 X 10 5 D value 1.9 ) Survivors – 0 min 250,000 – 1.9 min 25,000 – 3.8 min 2,500 – 5.7 min 250 – 7.6 min 25 – 9.5 min 2.5 – 11.4 min0.25 Complete kill is achieved in 10.7 minutes or 90 % of the 12 log reference cycle

75. Proof plus ( 1.3 X 10 4 D value 1.9 ) Survivors – 0 min 13,000 – 1.9 min 1,300 – 3.8 min 130 – 5.7 min 13 – 7.6 min 1.3 – 9.5 min 0.13 Complete kill is achieved in 8.7 minutes or 75 % or the 12 log reference cycle

77. Chemspor ( 2.5 X 10 2 D value 4.9 ) Survivors – 0 min 250 – 4.9 min 25 – 9.8 min 2.5 – 14.7 min 0.25 Complete kill is achieved in12.1 minutes or 102 % of the 12 log reference cycle

78. BI in overkill method With few exceptions, most commercial BIs do not verify the proposed 12 log reference cycle Most commercial BIs “pass” between 55 and 95 % of the reference cycle They won’t tell you if your sterilizer operate sub- optimally A “fail” tells you “Houston we have a problem “ Are not useful to tell where it is and how to judge its is important.

79. Chemical Indicators (CIs) Except for wax, most CIs are based on a pH change resulting from organic acid evaporation by heat and revealed by a colorimetric indicator The oldest CI Pb + S + H PbS The reaction do not occur without heat Paper CIs (chemical ink) are much more stable and predictable than CIs requiring assembly

80. Indicators : ISO Class 11140-1 Class 1 External indicator charged to signal if the pack has been exposed or not. Example : autoclave tape. Class 2Indicator for a specific parameter Qualitative Example : Bowie Dick (vaccum à 121 o C) Class 3Indicator for a unique parameter : Quantitative Example : Melting wax pellet at 121 o C

81. ISO 11140-1 Class 4Indicator sensitive to 2 parameters (ex.: time and temperature) within 25 % of expected targets. – For class 4 and higher, indicators must change abruptly – The change of color must happen within 25 % of the expected time and within 2 o C Example : An indicator that accept (OK or Pass) at 132 o C pour 4 min. MUST reject (Fail) a 130 o C for 3 minutes.

82. ISO 11140-1 Class 5An indicator/integrator sensitive to 2 or more parameters, and reacting within 15 % of expected targets. – In this category, the change of color must be abrupt, happens within 15 % of expected targets and MUST NOT happen if the targeted temperature is not achieved within 1 o C. Example : An indicator that accept (OK or Pass) at 132 o C for 4 min MUST reject (Fail) a 131 o C for 3 minutes and 22 seconds. Classe 6An indicator/integrator sensitive to 2 or more parameters, and reacting within 6 % of expected targets. – In this category, the change of color must be abrupt, happens within 6% of expected targets and MUST NOT happen if the targeted temperature is not achieved within 1 o C. Example : An indicator that accept (OK or Pass) at 132 o C for 4 min MUST reject (Fail) a 131 o C for 3 minutes and 45 seconds.

83. Enemy of steam sterilization : air Why, why, why For the same reasons we wash the plates in standing position (specialy with gravity cycles) (unwrapped material) Avoid stacking up Avoid “asparagus assembly” Question : Are you sure that things are sterile under the rubber bands

84. My daughter’s trick ?

85. Air displacement By gravity By dilution (flash) By pressure pulse By vacuum By pressure pulse and vacuum (Steam Flush Pressure Pulse) By high pulse pressure (Above Atmospheric Steam Flush Pressure Pulse)

86. Water, eau, H 2 O Water is a source of life and headaches Water is source or stains and deposits Hard water : contains a lot mineral salts and generally has a basic pH – calcium deposits, pipe crusting – A basic pH means less hydrogen ions availability Soft water: few mineral salts but is generally much more corrosive being most of the time much more acid.

87. Canada Red stain : iron Brown stain : Manganese, Magnesium Green or blue stain : copper Viscous and green “Stuff” : “metallothropic bacteria Hardness according to regions

88. Micro organisms found in potable water Divers viruses (Norwalk like, enterovirus) Pseudomonas (aeruginosa in particular) Acinetobacter spp Burkolderia cepacia Aeromonas Legionella non TB Mycobacteria Toxoplasma gondii Cryptosoridia

89. Water Surveillance Water quality requirements in hospital should be high because : – Immunosupressed patients – Types of treatments – Needs for cleaning, disinfection and sterilization

90. Unacceptable usages for tap water Endoscope rinsing after disinfection ; (Why ?) Washing of wounds and ulcers ; (Why?) Drug nebulizers ; (Why ?) Dialysis ; (Why ?) CSR nebulizers and ultrasonic baths : (Why?)

91. Question period

92. Is your brain also steaming with heat Thank you