ATP Adenosine TriPhosphate Bioluminescence. What do we ... · Agenda 1. ATP Bioluminescence...

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ATPAdenosine TriPhosphateBioluminescence.What do we need to know?

Decon Safety Prog Network Event

Dublin

26th March 2018

Dr Brian Kirk3M UK

Agenda

1. ATP Bioluminescence Technology – What it is and what it is not, how it works.

2. What are its characteristics and what benefits does it bring.

3. What standards say.

4. Applications areas

5. Endoscopy – why do we care about cleanliness. The clinical evidence.

6. Data reporting.

7. Questions

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• ATP is present in ALL living cells

• Microbes

• Plants

• Animals

• Humans - tissue and Bodily Fluids

• ATP stores energy in the phosphate bonds.

• Energy is released when the phosphate

bonds are broken

• This energy used in cell metabolism

Adenosine

Phosphates

Adenosine Tri-Phosphate

What is Adenosine Triphosphate (ATP)?The energy molecule for life

3M Confidential

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The principle of ATP bioluminescence

ATP:

Adenosine Triphosphate

The “energy currency” molecule of all living organisms

Luciferin/luciferase+

Indicates

contamination of

biological origin

BioluminescenceBioluminescence is where the energy of a biochemical bond (ATP to ADP) is converted to light energy via a biochemical reaction.

The reaction takes place in the cold and is found in many natural processes

Origin:

• Greek bios for “living”

• Latin lumen for “light”

Bioluminescence in nature

ATP - Adenosine triphosphate

The “battery” of all living cells.

Found in all living or once living biological cells.

Source of energy easily stored and used when needed for cellular functions

Cleavage Cleavage Cleavage Cleavage of phosphate from ATP releases energy of phosphate from ATP releases energy of phosphate from ATP releases energy of phosphate from ATP releases energy

ATP +HATP +HATP +HATP +H2222O O O O ADP ADP ADP ADP + HPO+ HPO+ HPO+ HPO33332222----

ΔΔΔΔЄ (30kJ/(30kJ/(30kJ/(30kJ/molmolmolmol))))

Bioluminescence - mechanism

The following reaction is catalysed by the enzyme luciferase and yields light:

*Light emitting bio pigment

ATP + D-Luciferin* + O2

Oxyluciferin + ADP + phosphate + CO2 + Light Photons (RLU’s)(560nm)

Luciferase

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increase in organisms and other biological residuesincrease in organisms and other biological residuesincrease in organisms and other biological residuesincrease in organisms and other biological residues

increase in ATP levelsincrease in ATP levelsincrease in ATP levelsincrease in ATP levels

increase in light (RLU)increase in light (RLU)increase in light (RLU)increase in light (RLU)

Simple Relationship

ATP Bioluminescent Intensity vs concentration A highly sensitive method

Copyright – Dr Virginia Deibel, TRAC Microbiology

attoM femtoM picoM

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The emitted light is detected by a luminometer

Light is converted into a number - RLU

– relative light units.

The bigger the number, the more ATP detected, the

“dirtier” the surface sampled

How the emitted light is measured.

Technical explanation:

Photons of light enter a photomultiplier tube and impact on a special crystal which releases electrons as a consequence of the photon excitation. The released

electrons enter a cascade of charged electrodes which release more electrons. The electron stream creates a current flow which is amplified. The amplified signal

enters an analogue to digital converter circuit. The digitised signal is further processed and finally presented on screen as a numerical value

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What Can ATP Bioluminescence tell us;

A very sensitive marker of biological contamination.

HOWEVER:

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How Do ATP Results Relate to Microbiology Methods?

ATP + Firefly enzyme = 200 RLU

CFU

RLU

With human residues, RLU does not correlate

well with CFU

Non-Microbial ATP

Microbial ATP

With these typical proportions of product and microbial

ATP on a swab RLU does not correlate with CFU but

with “cleanliness”

They Don’t except in pure microbial cultureThey Don’t except in pure microbial cultureThey Don’t except in pure microbial cultureThey Don’t except in pure microbial culture

ATP - Bioluminescence

Does not measure residual Does not measure residual Does not measure residual Does not measure residual proteinproteinproteinprotein

Very sensitive measure of residual tissue debris associated

with incompletely cleaned devices.

EN ISO 15883…

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A Log Normal function was found to provide

the best fit to the data (solid red line, R2=0.72;

95% confidence intervals shown as dotted red

lines). The analysis shows that ATP and protein

levels are linearly correlated (dashed black line,

R2=0.68) for protein concentrations greater

than 8 µg/mL and ATP values greater than 20

RLUs (corresponding to an ATP concentration of

~7 fmoles/mL)

The study measured levels of protein and ATP

in 89 endoscopes (48 colonoscopes, 38

gastroscopes, 3 duodenoscopes) at two US

clinical sites for three points during

reprocessing: bedside cleaning, post-manual

cleaning, and after high level disinfection

(HLD)

Relationship between residual protein and ATP in complex soils

Detection of Whole Blood – a complex soil

• Ten fold dilutions of whole blood• Determined the analytical limit of detection to be less than 1x10-7 diluted

whole blood (between 5-50 RBCs/swab)

Other tissues similarly detected.

ATP Sensitivity - Detection of Whole Blood

y = 0.9571x + 8.9202

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

-9-8-7-6-5-4-3-2

AT

P [

LO

G(R

LU

s)]

Whole Blood Dilution [LOG(dilution)]

Instrument Background

• Ten fold dilutions of whole blood

• Determined the analytical limit of detection to be 1x10-8 diluted whole blood (approximately

50 RBCs/swab)

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� A sensitive and objective method compared to visual assessment� A sensitive and objective indicator of generic soil (living or once living) – non specific.

� Results are available in less than one minute.

�Provides a definition for what “clean” is Provides a definition for what “clean” is Provides a definition for what “clean” is Provides a definition for what “clean” is ---- quantificationquantificationquantificationquantification

�Implement simple pass / caution / fail levels

�Gives chance to take corrective action – re-clean

� Monitor and understand hygiene levels over time – reveals variability and indicates consistency

ATP bioluminescence

What is meant by clean – in the past3.9 Cleaning:

Removal of contamination from an item to the extent necessary for its further processing and its intended subsequent use.

• EN ISO 15883EN ISO 15883EN ISO 15883EN ISO 15883----1:2006 1:2006 1:2006 1:2006 –––– Washer Disinfectors Washer Disinfectors Washer Disinfectors Washer Disinfectors –––– General requirements terms definitions and General requirements terms definitions and General requirements terms definitions and General requirements terms definitions and tests.tests.tests.tests.

So how clean should a re usable surgical instrument be that will be used in a body So how clean should a re usable surgical instrument be that will be used in a body So how clean should a re usable surgical instrument be that will be used in a body So how clean should a re usable surgical instrument be that will be used in a body cavity?cavity?cavity?cavity?

The alert and action level criteria for other analytes are:

What are the main methods available to assess cleanliness:According to ISO 15883-5 (CD)

What is “Clean” ?What is “Clean” ?What is “Clean” ?What is “Clean” ?

The alert and Action level criteria for protein assays are:Alert Level >= 3 microg/ cm2

Action Level >= 6.4 microg / cm2

AnalyteAnalyteAnalyteAnalyte Alert Level (per cmAlert Level (per cmAlert Level (per cmAlert Level (per cm2222)))) Action Level Action Level Action Level Action Level (per cm(per cm(per cm(per cm2222))))

ATP >10 femto moles >22 femto moles (1 x 10 – 15 )

Total Organic Carbon N/A >12 microg/ cm2

Carbohydrate N/A >1.8 microg/ cm2

Haemoglobin >1.0 microg/ cm2 >2.2 microg/ cm2

Endotoxin >2.2 EU/ cm2 N/A

How to test for cleanlinessEN ISO 15883EN ISO 15883EN ISO 15883EN ISO 15883----1 1 1 1 ---- 6.10 6.10 6.10 6.10 –––– Test of cleaning efficacyTest of cleaning efficacyTest of cleaning efficacyTest of cleaning efficacyTest 1 – apply heavy soil to load, wash then estimate cleaning efficacy visuallyTest 2 – using a normally contaminated load, wash then estimate cleanliness:• Visually• By a residual protein test

• Ninhydrin methodNinhydrin methodNinhydrin methodNinhydrin method

• OPA (Ortho OPA (Ortho OPA (Ortho OPA (Ortho phthallicphthallicphthallicphthallic dialdehyde) methoddialdehyde) methoddialdehyde) methoddialdehyde) method

• Biuret methodBiuret methodBiuret methodBiuret method

The sensitivity of each of these methods is different hence the level The sensitivity of each of these methods is different hence the level The sensitivity of each of these methods is different hence the level The sensitivity of each of these methods is different hence the level of cleanliness measured differs.of cleanliness measured differs.of cleanliness measured differs.of cleanliness measured differs.

What do we mean by “Clean”The definition of clean was largely driven by the sensitivity of the test methods used

• ie no absolute definition

Visually clean could mean significant quantities of tissue / blood remain with possible “infectivity”.

Standards specify residual soil (protein/cellular debris) test methods BUT varying sensitivity (see

later)

Research methods use ultra sensitive methods.

WHICH IS CORRECT ???

How effective are current cleaning methods using AWD’s

After UseAfter UseAfter UseAfter Use:Tissue load after use – estimated at 1 to 60mg or more tissueAfter cleaning and sterilizingAfter cleaning and sterilizingAfter cleaning and sterilizingAfter cleaning and sterilizing120 cleaned instruments analysed from 5 NHS hospitals. • Tissue Load After cleaning 0.2 to 0.7mg protein

• Baxter et al, (2006), J Hosp Inf, 63(3), 439-434

206 cleaned and sterilized instrument packs analysed for residual protein• 17% contaminated with between 3ug and 45mg of protein

• Murdoch et al, (2006), J Hosp Inf, 63(3), 432-438

23 instruments monitored for contamination using EDIC/EP microscopy• All instruments showed contamination, 56% gross (21 ug protein)

• Lipscombe et al, (2006), J Hosp Inf, 62, 141-148

Prion Infective dosePrion Infective dosePrion Infective dosePrion Infective dose:10 000 molecules, 0.6 x10-15 g (femtog) is one infective unit.

• Barron et al (2007),J Biol Chemistry, 49, 35878-86

Three Main Applications

Environmental SurfacesATP

Endoscopy monitoringATP

Surgical InstrumentsProtein (& ATP)

Some Examples of using ATP to assess cleanliness from the Literature

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• One hospital Italy• 3 WDs• 435 ATP results• 1.3 % above 200 RLU• Results showed

significant differences by• load pre-treatment, • washing program,• load material • load amount

• Suggesting new levels of transparency possible

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• 18 WDs in 8 hospitals in 4 European countries• Every load from each WD monitored for at

least one week• 1006 data points

• 64 negative controls, • 62 empty loads • 880 regular process runs

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Study Results• Median

• negative controls = 4 • empty loads = 5• regular process runs = 24

• Variation much higher on process loads 1 – 6578

• Differences in values for empty and regular loads were statistically significant

• 4% results >200 RLU4% results >200 RLU4% results >200 RLU4% results >200 RLU• 1% results > 500 RLU• These results identify outliers• Reveal detailed picture

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Study Conclusions

• Visual inspections of instruments after AWD processing does not provide full picture.

• The broad variation in RLU results suggest every load monitoring appropriate to ensure the end

result is satisfactory.

• ATP method is potentially valuable in the daily monitoring of WDs.

s

Endoscopy Monitoring

ECRI 2018 – Top 10 Health Technology Hazards

31

Contamination Rates & Soiling Levels

Recent studies suggest that 10-20% of flexible endoscopes contain high levels of viable microbes after reprocessing is complete….

Margin of safety

Rutala & Weber. JAMA 2014; 312: 1405-6

Microorganisms in the internal channel of gastrointestinal endoscopes = 8888----10 log10 10 log10 10 log10 10 log10

Reduction from the manual cleaning step in endoscope reprocessing = 4 4 4 4 ---- 6 log106 log106 log106 log10

Reduction from high-level disinfection steps = 4 4 4 4 ---- 6 log10 6 log10 6 log10 6 log10

___________

Total reduction of microbes = 8 8 8 8 ---- 12 log1012 log1012 log1012 log10.

Minimal margin of safety Minimal margin of safety Minimal margin of safety Minimal margin of safety in cleaning and high-level disinfection of GI endoscopes = = = = 0000----2 log10 2 log10 2 log10 2 log10

Approximate margin of safety associated with cleaning and sterilization of surgical instruments

= 17 log10 17 log10 17 log10 17 log10

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Recent endoscopy-associated CRE Outbreaks in US

The Scale of the Problem

Since 2012, >40 documented duodenoscope related

outbreaks involving Multi-Drug Resistant (micro)

Organisms with over 350 patient infections and >30

deaths have been reported1

1 Murray 2016 Preventable Tragedies: Superbugs and How Ineffective Monitoring of Medical Device Safety Fails Patients

Typical Endoscope Reprocessing according to guidelines

1.

Manual

cleaning

at

bedside

2

Transport

to

reprocess

ing room

3

Leak

testing

4

Manual

cleaning:

a. Immerse

b. Wipe

c. Brush

d. Flush

e. Rinse

f. Dry

5Automatic

Endoscope

Reprocessor:

6

Drying

&

Storage

Po

st A

ER

Po

st m

anu

al

Suction Biopsy Channel

Po

st b

edsi

de

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Process control of Endoscope Reprocessing with ATP

• Evaluate the use of ATP as process indicator for process control of cleaning ofendoscopes

• Monocenter Study

• 60 Endoscopes from daily routine

• 6 non-consecutive days

• Single reprocessing technician• 8 gastroscopes of one manufacturer• Guideline compliant reprocessing with enzymatic

detergents and gluataraldehyde

ATP Water TestBedside

Flush

•ATP Water Test

•Microbiology

Manual Cleaning

•ATP Water Test

•MicrobiologyAER

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Endoscope Specific Process Performance

1

10

100

1000

10000

100000

1000000

Vorreinigung Bürstenreinigung nach RDG-E

AT

P [

log

RL

U]

Parohl et al, GMS Hygiene & Infection Control, 2017, Vol. 12

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Endoscope Contamination per Process Step

nach RDG-EBürstenreinigungVorspülung

5

4

3

2

1

AT

P [

log

RLU

]

2,3

Boxplot: ATP Verschmutzung Log RLU

Parohl et al, GMS Hygiene & Infection Control, 2017, Vol. 12M.J. Alfa, I. Fatima, N. Olson; Am. J. Infect. Control; 41 (2013) 249.M.J. Alfa, I. Fatima, N. Olson; Am. J. Infect. Control; 41 (2013) 245.

FindingsFindingsFindingsFindings

ATP aftermanual cleaning>200 RLU>200 RLU>200 RLU>200 RLU 31/6031/6031/6031/60CFU after AER>10 CFU/10 ml>10 CFU/10 ml>10 CFU/10 ml>10 CFU/10 ml 9/609/609/609/60

7/9 Endoscopes with microbiological finding have had more than 200 RLU after manual cleaning

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Control Chart after Manual Cleaning

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140 160 180

AT

P [

RL

U]

Endoskop Messung

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Control Chart before and after AER

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140 160 180

AT

P [

RL

U]

Endoskop Messung

0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120 140 160 180

AT

P [

RL

U]

Endoskop Messung

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Conclusion

ATP Bioluminescence is a highly sensitive useful TOOL for monitoring for the presence of complex biological contamination likely to be found on inadequately cleaned surgicalinstruments or endoscopic devices.

Offering quantitative results it allows development of quality assurance programmes whichensure adequate cleaning of complex re-usable devices thereby improving patient safety.

Thank You