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
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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
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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”
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Bioluminescence in nature
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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))))
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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
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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
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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
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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.
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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
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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?
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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
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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.
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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 ???
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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
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Three Main Applications
Environmental SurfacesATP
Endoscopy monitoringATP
Surgical InstrumentsProtein (& ATP)
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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.
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s
Endoscopy Monitoring
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ECRI 2018 – Top 10 Health Technology Hazards
31
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Contamination Rates & Soiling Levels
Recent studies suggest that 10-20% of flexible endoscopes contain high levels of viable microbes after reprocessing is complete….
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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
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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
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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 [
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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
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Control Chart before and after AER
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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.
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Thank You