Asst. Prof. Dr. Mehmet İLKTAÇ -...

PHAR457

Asst. Prof. Dr. Mehmet İLKTAÇ Lecture 6: 25/12/2015

DEFINITIONS Disinfectant Chemicals used to kill harmful microorganisms or inhibit their growth on non-living surfaces and objects.

Ineffective against bacterial spores.

Antiseptic

Chemicals that can kill or inhibit growth of

microorganisms on living tissues like skin, oral cavities, and open wounds.

Not effective against spores.

TO BE LIVING OR NOT TO BE !!!

DIFFERENCE

DEFINITIONS Biocide Chemical agents that kill all living microorganisms including spores.

Biostat Chemical agents that inhibit microbial growth but do not kill microorganisms.

Sporicide

Chemical that kills all living microorganisms including spores.

Synonym to biocide

DEFINITIONS Germicide

Chemical agents that kills pathogenic organisms.

Not sporicidal.

Used on both living tissues and non-living objects

Antiseptics Disinfectants

Disinfection Process that reduces or eliminates microorganisms with the exception of spores.

PROCESSES THAT PROVIDE A 5 LOG (105) REDUCTION IN MICROBIAL CONTENT.

DEFINITIONS Sanitization

Process by which the bioburden of area is taken to a safe level.

Applied to substances used on inanimate substances.

PROCESSES THAT PROVIDE A 3 LOG (103) REDUCTION IN MICROBIAL CONTENT.

Disinfection: 5 log reduction

DIFFERENCE

DEFINITIONS Decontamination

Process by which the bioburden of area is removed.

Can be done by sterilization, disinfection, sanitization or mechanical processes.

DISINFECTANTS IN PHARMACEUTICAL INDUSTRY

Excellent cleaning and sanitization program is necessary

To prevent microbial contamination of sterile and non-sterile products

Pharmaceutical ingredients (2) Process water (1) Packaging components Manufacturing environment Processing equipment (3) Operators (4)

SOURCE OF CONTAMINATION To prevent contamination

Disinfectants

Antiseptics

FREQUENTLY USED DISINFECTANTS IN PHARMACEUTICAL INDUSTRY

Alcohols Phenols

Quaternary Ammonium Compunds Sodium Hypochloride

Peracetic acid Hydrogen peroxide Gluteraldehyde Formaldehyde Chlorine dioxide

Peracetic acid/Hydrogen peroxide

I. ALCOHOLS 70 % ethanol and 70 % isopropyl alcohol.

Act on cytoplasmic membrane and solubilize lipids, denature membrane proteins

DEHYDRATE CELLS

Absolute alcohol is not antimicrobial !!

NEED WATER FOR

ANTIMICROBIAL ACTICITY

Fast acting disinfectant ACTIVITY REDUCED BY ORGANIC MATERIAL

II. PHENOLS Phenol, irgasan, hexaclorophen, chlorocresol, chloroxylenol

Target sites cytoplasmic membrane, cell wall and intracytoplasmic coagulation

Microbicidal and microbiostatic

Activity reduced in alkaline pH and organic matter

Not compatible with QAC, iodophors and detergents

III. QUATERNARY AMMONIUM COMPOUNDS

Benzalkonium chloride

Cetrimide

Act on cell membrane, increase permeability and intracytoplasmic coagulation.

Cationic, surface active substance

Inhibited by anions, acidic pH and organic matter.

Incompatible with phenols, detergents and anionic compunds.

IV. SODIUM HYPOCHLORIDE

Act on cell wall, enzymes and amino groups

Weak sporicidal

Not compatible with hydrogen peroxide, detergents or organic matter.

Corrosive

Bleach

V. PERACETIC ACID

Sporicidal

Toxic and corrosive.

Not compatible with organic matter.

VI. HYDROGEN PEROXIDE

Bactericidal and fungicidal

Antiseptic at 3% concentration

Sporicidal at 30%.

Acts on enzymes and ribosomes

VII. GLUTARALDEHYDE 2% concentration Excellent disinfectant in

few minutes

Sporicidal in at least 10 hours.

Target sites Cell wall, enzymes, cogulation, amino grops

VIII. FORMALDEHYDE

Sporicidal

Liquid and gas

Highly toxic

Not compatible with temperature below 22 °C and

humidity below 60-80%.

Acts on cell wall, cell membrane, enzyme, ATP, ribosome, nucleic

acid, coagulation

IX. CHLORINE DIOXIDE Gas

Sporicidal at low concentration and room temperature

Non-carcinogenic, non-flammable

Incompatible with copper and uncoated alluminium.

X. PERACETIC ACID/HYDROGEN PEROXIDE

Disinfectant

Sporicidal 3 h

15 min Nontoxic, noncorrosive liquid

SPORICIDAL DISINFECTANTS

Peracetic acid

Gluteraldehyde

Formaldehyde

Chlorine dioxide

Hydrogen peroxide at 30%

Peracetic acid/hydrogen peroxide

HOW TO SELECT DISINFECTANT?

WHICH POINTS SHOULD BE CONSIDERED IN SELECTING

DISINFECTANT ?

POINTS TO CONSIDER DURING THE SELECTION OF DISINFECTANTS

Bioburden

The type and the number of microorganisms.

The higher the number; the more difficult for the disinfectant to be effective.

Resistance varies according to microorganism type (vegetative bacteria, spore, virus, Mycobacteria...)

Vegetative bacteria (Staph., Pseudo ..)

Enveloped viruses (HIV ..)


Surface

The type of surface may react with disinfectants.

Compatibility

Hypochloride Peracetic acid

Corrosive

Organic matter Organic material like blood, soil, clinical material of the patient negatively affect performance of some disinfectants.

Alcohol, phenol, QAC, bleach, peracetic acid

Clean the surface before

Chlorine dioxide Copper and alluminium

Contact time

The time required for the disinfectant to be effective.

Must make the surface exposed to disinfectant for at least contact time period.


Disinfectants should be choosed considering their contact time and the time that the surface/device should be ready.

Goal of the process (Endpoint results)


I. TO MAINTAIN THE MICROBIAL POPULATION AT THE CURRENT LEVEL OR TO ENSURE THAT THE CONCENTRATION OF MICROORGANISMS DOESNOT INCREASE SIGNIFICANTLY

USE A DISINFECTANT THAT HAS STATIC EFFECT

II. TO SIGNIFICANTLY REDUCE MICROBIAL POPULATION

USE A DISINFECTANT THAT HAS CIDAL EFFECT

CIDAL VERSUS STATIC ACTIVITY


Preparation of disinfectant

Dilute the disinfection accurately to use-dilution to get desired result.

Generally diluted with water.

Dilute using aseptic thecniques and sterile purified water.

If not, sterilize with filtration

Choose disinfectants that are easy to prepare.


Shelf life Concentrate of a disinfectant Shelf life

determined by manufacturer

Shelf life of concentrate ≠ Shelf life of diluted disin.

Dilution affects the properties and stability of compund

It is user’s responsibility to determine the shelf life of diluted disinfection


Safety and Residues

Is the disinfectant safe ?

Inflammable ?

Toxic residues ?

Explosive ?

Remove

Use combined disinfectants

BE CAREFUL ABOUT

COMPATIBILITY

Phenol QAC

Phenol iodophore

QAC detergent

TESTS FOR THE EFFICACY OF DISINFECTANTS

1. Suspension tests

2. Use-Dilution test

3. Surface Challenge Tests

4. In situ tests

Companies who register a public health disinfectant must ensure the safety and effectiveness of their product

It is the user’s responsibility to provide evidence that the disinfectant is effective at reducing the microbiological load of environmental isolates on the surfaces found in their facility.

Inappropriate use, ineffective cleaning and ineffective disinfectants

Affect the quality of pharmaceuticals manufactured

in the environment

DISINFECTANT EFFICACY TESTS


1. Suspension test A sample of the bacterial culture is directly suspended into the disinfectant solution.

After contact time, a sample from suspended disnfectant is transferred to agar containing neutralizant

Qualitative or quantitative


1. Suspension test


1. Suspension test Quantitative Suspension method

Number of surviving microorganism is compared to initial inoculum.

loginoculum - logrecovery

Log reduction must be equal to or greater than log5.

Acceptance criteria

Suspension test is used as a simple screening to determine the type of disinfectant most effective against a specific set of organisms before performing a complete (full) disinfectant qualification.

Advantage Bacteria are uniformly exposed to the disinfectant


1. Suspension test

Disadvantage Not related with ‘’real life’’ disinfection process (no surface)

Use of Suspension test ?

USP doesnot imply suspension tests.

Use-dilution tests Surface challenge tests.

For disinfection efficacy testing, USP recommends

DISINFECTANT EFFICACY TESTS



To determine the effectiveness of the disinfectant at specific concentrations and exposure times by inoculating the specific concentrations of disinfectant with the determined organisms.



Carriers Stainless steel cylinders

Transferred into suspensions of test culture

Dried in incubator

Transferred into tubes containing disinfectants

After contact time transferred into broths containing neutralizant

Incubate 37 C, 48 h

1. The carrier is contaminated by submersion in a liquid culture of the test organism.



2. The carrier is then dried and is brought in contact with the disinfectant for a given contact time.

3. Transfer the carrier into broth containing NEUTRALIZING AGENTS.

4. No growth (turbidity) indicates activity of the disinfectant; whereas turbidity indicates failing.

Salmonella S aureus P. aeruginosa



Test strains

Acceptance Criteria 10 out of 10 carriers negative

59 out of 60 carriers negative

No turbidity in the broth

In addition to test strains frequently isolated ENVIRONMENTAL

STRAINS should also be tested.

LIMITATIONS OF USE-DILUTION TEST

1. The number of bacteria dried on a carrier is hard to standardize

2. The survival of the bacteria on the carrier

during drying is not constant.

3. It does not mimic the real life disinfection method.

In real life disinfection, the surface is not submerged into disinfectant. Disinfectant is sprayed or mopped or wiped onto surface.


3. Surface Challenge Tests Surface challenge testing involves the creation of a dried organism film onto representative surface types.

Surface chalenge tests both mimics the contaminated environment and the disinfection procedure performed in facility

Imitates ‘’real life’’ disinfection procedure

Coupons Representatives (miniatures) of the actual facility surfaces that are disinfected

5 cm x 5 cm



Plastic, metal, glass, rubber, latex, vinyl, aluminium, steel

Choose the coupon that represents the surface to be disinfected



Challenge (test) organisms

Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa Candida albicans Aspergillus brasiliensis Bacillus subtilis

+ ENVİRONMENTAL STRAINS

Gr (+) coccus

Gr (-) bacilli

Spore forming

Fungi

Surface Challenge Tests Most frequently

isolated


3. Surface Challenge Tests – Steps 1 1. Prepare challenge organisms and environmental

organisms

2. Inoculate coupons with each of the challenge and environmental organisms.

3. After the coupons have dried, treat them with disinfection.

Treat the coupons with disinfectant using the same application method that you will use in the ‘’real life’’.

To make the procedure closest to real procedure



during application of disinfectant to coupons, use the same method


3. Surface Challenge Tests – Steps 2 4. After treating the coupon with disinfectant, wait

for contact time

In order to determine the best contact time;

Use the recommended contact time One or two times below the recommended time One or two times above the recommended time

t= 2, 4, 10, 12, 15m t= 5, 10, 15m 1 time above and below 2 times above and

below

Recommended

The time during which the disinfectant does its job.


3. Surface Challenge Tests – Steps 3 5. Take samples from coupon that is treated with

disinfectant and test strain and from coupon that is untreated with disinfectant but treated with test strain

Sampling Methods

Swabbing Contact plates

Surface rinse

Flat surfaces

Irregular surfaces

Samples should be processed in the laboratory immediately.

If more than 60 min; refrigarate the sample.

Swabs must be moistened before taking the sample and remain moistened through the holding time.

Use transport media

Prevent both the drying and proliferation of microorganisms.

Phosphate buffer Ringer solution

6. Determine the number of cfu/ml of disinfectant treated and untreated coupon by plate count methods.


3. Surface Challenge Tests – Steps 3

The media that the samples (swaps ) are streaked onto or contact plates must contain neutralizer.

Determine log reduction by subtracting logbeforefrom logafter

logbefore - logafter

Treated coupon Untreated coupon

Disinfectant Neutralizers in Media

3. Surface Challenge Tests – Steps



Acceptance criteria

2 log reduction bacterial spores 3 log reduction vegetative

bacteria, yeast, mold spores


4. IN SITU TESTS After evaluating the efficacy of disinfectants under

laboratory conditions;

Perform the tests in real areas and on equipments to be routinely disinfected.

In sıtu

tests

Perform bioburden determination before disinfection, do disinfection and determine bioburden again.

Show lower count after disinfection


4. IN SITU TESTS Before and after in situ tests are not performed

routinely after the disinfection method has been validated.

If during environmental monitoring new

microorganisms are isolated or increase in the number of

microorganisms is detected, it must be carried out before and

after disinfection.

IMPORTANCE OF ENVİRONMENTAL

MONITORING

As part of an environmental monitoring program, comparison of the frequency and number of organisms recovered would be performed within the actual facility.

If a new organism is routinely being recovered

If not, test disinfectant efficacy against this microor.

determine whether or not this type of organisms was covered in the disinfectant efficacy.

If a new disinfectant is introduced into the cleaning regimen, efficacy tests should be repeated.

Asst. Prof. Dr. Mehmet İLKTAÇ -...

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