Sterilization and validation

Richard Marchand MDMedical Microbiologist and Infectious DiseasesAssistant 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 Process validation Biological Indicators Chemical Indicators Water, water, water…

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.

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

Steam sterilization origins

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

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.

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 concept

using heat resistant spores

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

How does heat kill micro organisms ?

Heat coagulates proteins : egg white Heat has a mild oxidative effect

What is oxidation ?

Will discuss that later

Water Boils at

100 °C : fresh water in a pan with a lid 97 °C : fresh water in a pan without a lid104 °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 )

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)

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.

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.

SURVIVAL PROBABILITY

How to define resistance to heat ?

The D value concept was invented !

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.

Effect of probability for 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

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 to 7.5 min)

100 cans/100 BI (2.0 X 108) = 20 surv. (or to 8.5 min)

103 cans/103 BI (2.0 X 109) = 200 surv. (or to 9.5 min)

104 cans/104 BI (2.0 X 1010) = 2000 surv. (or 10.5 min)

105 canss/105 BI (2.0 X 1011) = 20000 surv.(or 11.5 min)

More we have cans, more we have contaminated cans !

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)

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

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

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.

HISTORY cont.

1960-70 Development of the Dvalue concept

using 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

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.

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.)

Little quizzz

Question : Is all processes D values a time dependant measure ?

Answer : No, for radiation and ozone sterilization the Dvalue is dose dependant

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

SURVIVAL PROBABILITY

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

STERILITY ASSURANCE LEVEL

Effect of probability for 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

Effect of probability for many packs

Cycle of 6.5 minutes 1 pack/1 BI (2.0 X 106) = 0-0.2 survivor 10 packs/10 BI (2.0 X 107) = 2 survivors (or to 7.5 min)

100 packs/100 BI (2.0 X 108) = 20 surv. (or to 8.5 min)

103 packs/103 BI (2.0 X 109) = 200 surv. (or to 9.5 min)

104 packs/104 BI (2.0 X 1010) = 2000 surv. (or 10.5 min)

105 packs/105 BI (2.0 X 1011) = 20000 surv.(or 11.5 min)

More we have packs, more wont pass !

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

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

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

Effect of liquids or heat barriers

Quizz

Question : Can you name a heat barrier material ?

Answer : polymers (plastics) like non porous plastic containers, wood, rubber etc..

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 orthopaedicsteel and metallic devices

In the real . . . Pre conditioning

Sterilization 3.5 to 15 min

Dry time 15 to 20 minutes max.

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

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.

Little quizzzzz :

Name the critical parameters of steam sterilization

Temperature (121 oC) Time (duration) Humidity Pressure (includes vacuum) Phases of cycle (manufacturer and Europe)

134oC 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.

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

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 ?

A lot of stored energy !

Can water exist as a gas at temperature lower Can water exist as a gas at temperature lower than 0than 0ooC ?C ?Can water not be steam at temperature higher Can water not be steam at temperature higher than 100than 100ooC ?C ?

Yes, humidity

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.

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”

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)

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

It takes 538 calories to convert one mL of water at 100oC to steam at 100oC

100 200 300 400 500 600 700water

Low energy vapour

High energy vapour

STEAMT

E

M

P

Instruments cause a temperature drop.

100 200 300 400 500 600 700

One or two degrees of temperature drop here…

Releases hundreds of calories of heat here.

The energy contained and released is called : HEAT OF CONDENSATION

100 200 300 400 500 600 700

To condense or not to con dance

100 200 300 400 500 600 700

If to much energy is released the steam goes back to liquid water ( full condensation) = the devices will be wet.

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)

100 200 300 400 500 600 700

A very fine tuning line to control

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)

Steam quality is important

Saturated steam 98% steam, 2% water vapor

Dry steam Superheated

Wet steam Supersaturated

STERILISATION VALIDATION

Sterilisation validation is arbitrarily laid on the

construction of an cycle based on the

behaviour of biologic indicators hopped to be

« predictable »

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)

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

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

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

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 $$$$$)

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.

3M Rapid Indicator

3M Rapid Indicator

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

Sportrol (1.4 X 105 Dvalue 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)

Attest (2.5 X 105 Dvalue 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 min 0.25

Complete kill is achieved in 10.7 minutes or 90 % of the 12 log reference cycle

Proof plus (1.3 X 104 Dvalue 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

Chemspor (2.5 X 102 Dvalue 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

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.

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

Indicators : ISO Class 11140-1

Class 1 External indicator charged to signal if the pack has been exposed or not.

Example : autoclave tape.

Class 2 Indicator for a specific parameter Qualitative Example : Bowie Dick (vaccum à 121

oC)

Class 3 Indicator for a unique parameter : Quantitative Example : Melting wax pellet at 121 oC

ISO 11140-1

Class 4 Indicator 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 oC Example : An indicator that accept (OK or Pass) at 132oC pour 4

min. MUST reject (Fail) a 130oC for 3 minutes.

ISO 11140-1

Class 5 An 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 1oC. Example : An indicator that accept (OK or Pass) at 132oC for 4 min MUST reject  (Fail) a 131oC for 3 minutes and 22 seconds.

Classe 6 An 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 1oC. Example : An indicator that accept (OK or Pass) at 132oC for 4 min MUST reject  (Fail) a 131oC for 3 minutes and 45 seconds.

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

My daughter’s trick ?

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)

Water, eau, H2O

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.

Canada

Red stain : iron Brown stain : Manganese, Magnesium Green or blue stain : copper Viscous and green “Stuff” : “metallothropic

bacteriaHardness according to regions

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

Water Surveillance

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

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?)

Question period

Is your brain also steaming with heat

Thank youThank you