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Rachel Scott Unit 11 Wenta Business Centre Colne Way Watford Hertfordshire WD24 7ND

Manchester PCT Mauldeth House Mauldeth Road West Chorlton Manchester M21 7RL Tel: 0161 958 4003 Fax: 0161 958 4040 Email: peter.kevan@manchester.nhs.uk

27th March 2009

We at Manchester PCT use the Hygiena systemSURE to prove that our high standards of cleaning have been met in our Health Centre, Clinics and Care homes. The Hygiena systemSURE is simple to use and provides powerful information for our Facilities Team, Site Managers and of course our cleaning contractors. We generate reports for our infection control team and trust board. It is cost effective, robust and reliable and the extensive support and training we have received has been excellent.

Yours sincerely

Peter Kevan Contract Manager NHS Manchester PCT

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New technologies, monitoring practices gain traction

ARTICLE BY GINA ROLLINS DATA BY SUZANNA HOPPSZALLERN PHOTOGRAPHS BY CARROLL FOSTER


he important link between environmental cleanliness and infection prevention has long been appreciated, but how best to achieve these objectives remains a source of ever-changing science and application. So how are hospitals faring in this all-important area and how are they responding to the rapid changes in cleaning technologies, processes and verifying proper cleaning procedures are followed? More than four out of five are

2009 Infection Prevention + Hospital Cleaning Survey


either already using or planning to use advanced cleaning technology such as microfiber mops. Nearly one in four are augmenting observation-based audits and performance goals with newer technologies such as rapid environmental testing with chemical markers that fluoresce with ultraviolet light, or through environmental cultures. And many are turning to environmentally friendly cleaning products, even as theyre requesting evidence-based data to confirm the value of these products. These are among the many findings of a recent survey of environmental services managers and infection preventionists. The online survey was co-sponsored by Health Facilities Management and Materials Management in
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John Scherberger, director of environmental services, pastoral care and guest services at Spartanburg (S.C.) Hospital for Restorative Care, is an advocate for teamwork between environmental services and infection prevention.

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SPECIAL REPORT

How clean is CLEAN?

2009 INFECTION PREVENTION + HOSPITAL CLEANING SURVEY


lectual capital and collaborative efforts between ASHES, APIC and other organizations will better position our appeal for improved staffing ratios, widely accepted staff competencies and health care cleaning/disinfection protocols, training, education and quality assurance as well as sound science behind the claims of new products and technologies. The connection between environmental services and infection prevention is important in evaluating new products, training and educating staff about good hygiene practices, and even in eliminating prodThoroughly cleaning such items as monitors and TV remotes helps prevent health care-associated infections. In the far right photo, a cleaning agent is applied to a microfiber cloth that is then used to clean, which is proper cleaning protocol. ucts or procedures. That was the case at St. Health Care magazines, along with the American Society for Joseph Mercy Hospital in Ann Arbor, Mich. Our staff asked to Healthcare Environmental Services (ASHES) and the Associastop using disinfectant on the floors because they were develoption for Professionals in Infection Control & Epidemiology ing a cloud on the surface and environmental services was hav(APIC). The survey underscored that close collaboration being to strip and clean them more often, reports Russ Olmstead, tween environmental services managers and infection prevenepidemiologist. We did a literature review and found no evitionists is essential to achieving and monitoring performance dence of increased health care-acquired infections from using destandards for cleanliness of the patient environment, and for tergents instead of disinfectants on the floor, so we allowed them introducing new technologies and practices. to make the change. The updated Centers for Disease Control Its really a team effort. We cant operate in silos, says John and Prevention (CDC) Guideline for Disinfection and Sterilization Scherberger, director of environmental services, pastoral care in Healthcare Facilities, which can be accessed at www.cdc.gov/ ncidod/dhqp/pdf/guidelines/Disinfection_Nov_2008.pdf, also and guest services at Spartanburg (S.C.) Hospital for Restorative acknowledges that the use of germicidal chemicals to disinfect Care. I have an excellent working relationship with our infechospital floors and other noncritical items is controversial. tion preventionist and nurse managers. The good working relationship between infection prevenMonitoring cleanliness tionists and environmental services showed all the way through Observation-based audits continue to be the mainstay for the [respondents] comments, obmonitoring compliance with cleaning standards, with nearly serves Judene Bartley, vice president 87 percent of respondents using that method. However, many of Epidemiology Consulting Services health care institutions are finding ways to tighten observation in Beverly Hills, Mich. Bartley was protocols. For instance, Brigham and Womens Hospital in the APIC-appointed expert for the ealth Facilities ManageBoston maintains an electronic database of all rooms that survey. ment, Materials Manageenvironmental services supervisors access with handheld The survey data supports what ment in Health Care, the devices. This helps achieve the quality assurance goal of two ASHES has long believed, says Patti American Society for Healthinspections per employee per month. Costello, ASHES executive director. care Environmental Services When a supervisor takes the handheld, it shows all the emEnvironmental services and infec(ASHES) and the Association ployees what that person is responsible for and randomly identition preventionist professionals are for Professionals in Infection fies the rooms to be inspected so we dont have a selection bias, essential partners in providing a Control and Epidemiology explains Richard Bass, director of environmental services. The clean, safe, comfortable and high(APIC) teamed up to conduct system also has a results reporting feature that can provide sumquality patient care environment. the Infection Prevention & maries by employee, supervisor, time period and item inspected. Pathogenic microorganisms are Hospital Cleaning Survey. Other respondents couple observation-based audits with demore resistant and persistently presA random sample of tailed check lists to set and measure performance expectations. ent in the environment, staffing 3,538 infection preventionPinckneyville (Ill.) Community Hospital is an example. I develbenchmarks for infection preventionoped a task list that covers basically every item and surface in the ists were contacted to find ists and front-line cleaning techniout what steps hospitals are room. They vary depending on whether a patient is in the room cians are problematic, the Centers taking to optimize cleaning during the cleaning, staying in the room but not present during for Medicare & Medicaid Services protocols and reduce hospithe cleaning, or discharged, says Kevin Daugherty, environmen(CMS) and insurers are redefining tal services manager. We use this for our performance improvetal-acquired infections. The reimbursement and dramatic health ment standards and supervisors sign off on the lists when they survey response rate was care reform is on the horizon, check each room. 19.7 percent or 696 comCostello adds. The combined intel-

About the survey

pleted surveys.
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Top six ways hospitals measure compliance with cleaning standards in patient care areas
Observation-based audit

Cleaning practices and technologies hospitals routinely employ to disinfect patient rooms
Sodium hypochlorite, household bleach 68% 6% Quaternary ammonium disinfectant 85% 1% Disinfectant-impregnated wipers 77% 3% Microfiber cloths 46% 17% Microfiber mops Currently use 68% 14% Planning to use Copper and copper-alloy fixtures 4% 2% Pour bottles to dispense disinfectant 42% 4% Change cubical curtains after discharge of patients placed under contact precautions 57% 13% Hydrogen peroxide vapor decontamination system 2% 5%

87%
Patient satisfaction scores on cleanliness of room 78% Monitor compliance with performance targets for patient area cleaning 34% Risk-based audit 15% Environmental culture results 14% Measure cleaning rates of high-risk objects in patient areas 14%

Step hospitals have taken to optimize environmental services staff performance


Hands-on training in cleaning protocols 84% Education on transmission of health care-associated pathogens and resultant infection 81% Ongoing performance feedback 62% Predefined performance targets for patient area cleaning 31% Patient interviews by supervisory staff 27% Well-defined quality management program for patient area cleaning 24% Use of visually observable feedback tool (e.g., black-light marker, ATP) 20% Quality control assessments tied to compensation 10%

Top 11 challenges to cleaning and disinfection of the patient environment


Pressure to expedite room turns for incoming patients 42% Assigned responsibility for cleaning mobile objects 41% High hospital occupancy 35% Inadequate time to properly clean patient rooms and care areas 32% Reluctance to clean electronic equipment with saturated cloths 32% Inadequate staffing levels 31% Too busy/insufficient time allowed to consistently follow protocols 28% High turnover rates among environmental services technicians 26% Inadequate financial resources to invest in cleaning technologies and equipment 26% Lack of objective microbiologic standards for hospital cleaning 20% Lack of knowledge of the role specific high-risk objects play in transmitting health care-associated pathogens 20%

Hospitals using chemicals (e.g., ATP, fluorescing markers) to verify cleaning of the following high-risk objects
16% Bed rail 16% Tray table 16% Nurse call device 15% Bedside table 15% Bathroom doorknobs 15% Toilet seat 15% Patient telephone 14% 14% 14% 14% 13%
Sinks Toilet handle Patient room doorknobs and cabinet pulls Bathroom light switch Restroom grab bars

SOURCE: MMHC/HFM/APIC/ASHES 2009 INFECTION PREVENTION & HOSPITAL CLEANING SURVEY

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Nearly a quarter of the participants reported augmenting observation-based audits and performance goals with newer technologies such as rapid environmental testing with chemical markers that fluoresce with ultraviolet light, or through environmental cultures. The introduction of chemical markers in particular can have a profound effect on cleaning standards and protocols, according to Bartley. Its a more objective way of determining how well cleaning is taking place, and it can be very dramatic. Its a shock to find out that things you thought were clean in reality are not, she explains. These products are just beginning to gain traction in health care, Bartley adds, and it will be interesting to track their use and impact on cleaning in future surveys. (See chart on page 25.) Just how quickly this area of infection prevention is changing was evident during the Society for Healthcare Epidemiology of Americas (SHEA) annual scientific session in March. Results of a federally funded study were presented, concluding that risks of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) transmission could be lessened by immersing cleaning cloths in cleaning solution, educating workers and providing feedback on the removal of intentionally applied marks visible only under ultraviolet light. Regardless of the specific compounds used, these visually observable feedback tools make for excellent training tools, particularly with staff who may not be fluent in English. It immediately sends a message, no matter what language the individual is proficient in, that what they thought they removed is still there, notes Bartley. that they already use or plan to use microfiber mops, while 63 percent either already use or plan to use microfiber cloths (see chart on page 25). Organizations that have switched report multiple benefits. Spartanburg Hospital, a long-term acute care facility, transitioned to microfiber mops and cloths about four years ago. Since then, weve had no workers compensation injuries, no slips and falls from wet floors, and our waste-water treatment has decreased by hundreds of thousands of gallons, says Scherberger. Other survey respondents also gave the technology a thumbs-up. It saves labor because it picks up more. The employee still uses a figure-eight motion, but you get a cleaner sweep, explains Bobby Dorsett, director of environmental operations at Scripps Memorial Hospital La Jolla (Calif.). Microfiber products also minimize cross-transmission of microbes among patients, according to Cecilia DeLoach Lynn, senior manager of sustainable operations for Practice Greenhealth, a membership and networking organization for institutions committed to eco-friendly practices. Because they dont require re-dunking in a mop bucket and using the same water or mop for two or three rooms without changing, they really cut down on cross-contamination, she says. Copper or copper-alloy fixtures, a technology touted for its antimicrobial properties, has yet to gain traction among survey respondents. Less than 4 percent reported using the fixtures now, and nearly 94 percent indicated they have no plans to implement the technology. In laboratory studies copper has been shown to have antimicrobial properties, but its a challenge to take lab findings and apply them in a natural health care environment, observes Olmstead. Its an interesting technology thats not quite ready for prime time.

Adopting new technologies


The survey also revealed that many hospitals are incorporating the latest technologies to achieve and monitor performance standards for cleanliness of the patient environment. For example, more than 80 percent of respondents indicated

On going green

Putting green in perspective


s hospitals seek ways to transition to environmentally preferred practices and solutions, teamwork between infection preventionists and environmental staff must come to the fore, according to Cecilia DeLoach Lynn, senior manager of sustainable operations for Practice Greenhealth, an organization promoting eco-friendly practices. They need to look at effectiveness first before putting on the green filter, she says. Being sure of all the data and evidence takes close collaboration. Lynn is encouraged by the responses on environmentally preferred practices in the Infection Prevention & Hospital Cleaning Survey, particularly the use of microfiber mops and cloths. Weve seen that technology really take hold in a lot of facilities, she says. Its a win-win for infection prevention and the environment.

Practice Greenhealth encourages the use of products that have been certified as green. We recommend third-party certification of green cleaners because it holds the product to a standard and worker safety is ensured, she explains. We recognize that it might be difficult for small companies in the innovation stage to afford the cost of obtaining green certification, but unfortunately others will slap a green label on a product thats not really green. Two organizations that certify health care products as green include Green Seal in the United States and the EcoLogo program based in Canada. For facilities seeking to adopt environmentally preferred technologies, more information is available through the Web site of the Green Guide for Health Care at www.gghc.org. I

Hospitals are beginning to implement environmentally preferred cleaning initiatives, but the health care industry is advancing cautiously for good reasons, according to Bartley. Weve been slower to adopt the products because we know there are certain chemicals we have to use, she says. Until there are safer materials and more science behind their use, were left with infection preventionists doing risk assessments to determine where its safe to use [environmentally friendly] materials and where its required to use harsher ones. (See sidebar at left.) The most common environmentally preferred actions among survey respondents included orientation and training about the hazards, use and disposal of cleaning products (64 percent), policies on limiting exposure to chemicals (54 percent) and on environmentally preferable cleaning (57 percent), use of pre-diluted disinfectant systems (53 percent) and use of equipment that doesnt negatively impact indoor air quality (52 percent). At Brigham and Womens Hospital, the environmental safety department vets all new products, and use of the more toxic chemicals is limited to staff members with special training. In addition, the hospital uses fewer products than in the past. The vast majority of our staff is of low skill-level

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and weve dropped the number stand the reasoning behind of products we work with to it, you wont get the effort about five or six versus dozens needed, notes Daugherty. Ive seen used in other hospiHis staff receives ongoing, tals, Bass says. Our bottles are detailed education about the color-coded and bilingual. source of microbes and what Like Brigham and Womens, it takes to clean surfaces apmany facilities reported streampropriately. At Spartanburg lining the number of products Hospital, environmental used and minimizing worker services employees are asexposure to chemicals. signed to specific units and For instance, Spartanburg attend staff meetings for Hospital for Restorative Care those areas. Theyre part of now uses only four products, the team, and that provides and all are green-certified except positive reinforcement, the disinfectant. All our chemiScherberger explains. EnviJohn Scherberger, director of environmental services, pastoral care and cals are in a dispenser and envi- guest services at Spartanburg (S.C.) Hospital for Restorative Care, trains ronmental services has to ronmental services staff does not understand theyre just as two environmental services personnel on how to test for cleanliness. get involved in the mixing, says important as any other part Scherberger. They press one button for a lot of the chemical, of the team. Were the first line of defense in infection control. another for less, and I can check that their bottles are the propTesting for cleanliness er dilution based on the color. We also stopped using trigger The survey also revealed that the use of rapid environmental spray bottles. Theyre all pull-top so housekeepers saturate testing with substances that fluoresce under UV light is just their cloths rather than spraying in the environment. starting to be adopted in health care. However, experts predict Some facilities, including Curry General Hospital in Gold that adoption will become widespread soon. Beach, Ore., have made the switch to all sustainable products. While some technologies, such as adenosine triphosphate We already used products from this distributor, so we researched their green products to make sure they killed MRSA and the like and would not be irritating to our staffwe have a lot of people with allergies, explains Kim Sharp, environmental services manager.

Challenges and opportunities


Even as hospitals have made strides in implementing new technologies and practices, they still face challenges in achieving performance improvement objectives. Of respondents, 42 percent cited high hospital occupancy rates and the need to expedite room cleaning for new patients as major challenges to cleaning patient care areas, and they have employed a number of mechanisms to address this ongoing dilemma. St. Joseph Mercy Hospital redeployed environmental services staff from nonpatient care areas, and asked staff in those locations to pick up the slack by taking on tasks normally left to environmental services such as emptying trash and recycling bins, according to Olmstead. Scripps Memorial uses a teletracking system to keep nursing, environmental services, emergency and bed control staff up-todate on the status of rooms. It gives everyone information on unoccupied beds in real time, says Dorsett. When a bed becomes unoccupied, nursing staff punch in codes via the telephone, which sends a page to appropriate staff. Environmental services staff use the same system to notify other departments when they begin and end the cleaning of each room. This way, everybody at any given moment knows how many rooms are available, and how many are in the process of being cleaned. It helps with the anxiety around turnaround times, he explains. Perhaps the most crucial element of achieving performance standards for cleanliness of the patient environment is the overall approach taken in training and supporting environmental services staff. People can be trained to mop a floor, but if they dont under-

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(ATP) bioluminescence meters, have been used for years to detect microbes in the food industry, the science behind them has only been applied recently to the patient care setting. A leader in the field is Philip Carling, M.D., clinical professor of medicine at Boston University. Carling sought a way to improve on standard methods of verifying the cleanliness of patient care areas, primarily observation-based audits and less commonly, culturing of selected surfaces. He came up with a mixture that people jokingly refer to as Carlings Goo, which is a transparent, easily cleaned, environmentally stable marking solution that fluoresces when exposed to UV light. Carling and a consortium of participating hospitals have demonstrated in a series of studies that certain high-risk objects such as tray tables, toilet handles, bathroom light switches and bathroom and room doorknobs typically are not wellcleaned. Research shows that out of 14 high-risk objects, the lowest mean rates of cleaning were found for toilet doorknobs and toilet handholds (both 28 percent), bedpan cleaners (25 percent), room doorknobs (23 percent) and bathroom light switches (20 percent). Our findings are consistent that, on average, critical objects are only being cleaned about 50 percent of the time, and that there are several objects that are almost universally overlooked, Carling explains. Weve uncovered a systemic problem with the thoroughness of environmental health in hospitals because there was not a system for simple measurement in the past. Subsequent research has shown that with a focused performance improvement initiative, including education and feedback to staff, the average rate of cleaning can be increased from less than 50 percent to about 77 percent. Once the cleaning staff understands theyre critical to patient safety and are shown theyre not doing well in certain areas, thats when the paradigm shift occurs, says Carling. Use of a solution like Carlings can be an eye-opener even for facilities that have overall high rates of cleaning compliance, like Nebraska Medical Center in Omaha, which participated in Carlings studies. The program is fantastic. Its helped us identify various opportunities for improving our processes, says Mark Rupp, M.D., professor of infectious diseases at the University of Nebraska and director of epidemiology at Nebraska Medical Center. We found there were objects and surfaces that no one was taking responsibility for cleaning. Many were electronic such as a computer mouse. This enabled us to go item-by-item and figure out who would be responsible for cleaning them. Rupp also is president of SHEA. The utility of fluorescent marking is obvious even to facilities just beginning to use the solutions such as Scripps Memorial. Thats really exciting news, and its going to revolutionize the industry, Dorsett says. Well be able to tell whats clean and whats not. HFM
Gina Rollins is a Silver Spring, Md.-based freelance writer who frequently covers health care topics. Suzanna Hoppszallern is senior editor of data and research for Health Facilities Managements sister publication, Hospitals & Health Networks.

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Cleaning agents Clothing and PPE Construction Containment Decontamination Furniture and Equipment Healthcare Hospital acquired infections HVAC Hygiene Medical devices Microbiology Nanotechnology Pharmaceuticals Regulation Sterilisation Can the health sector learn from the food industry when it comes to improving hygiene? Professor Chris Griffith, head of the Food Research & Consultancy Unit at the University of Wales Institute, argues yes Healthcare associated infections (HCAIs) and the role of cleaning in their prevention have, and continue to receive a high level of media attention. However, the link between environmental cleanliness and HCAIs is the subject of debate.1,2,3 The UK guidelines for infection control 4 recommend that the hospital environment must be visibly clean with routine bacteriological sampling rarely indicated unless there is an outbreak of infection.5 The rationale behind this approach is based on the assumption that the inanimate hospital environment is of little importance in the spread of endemic infections but may occasionally have a role in outbreaks.5 This belief and approach is in direct contrast to views held by the food industry, which believes the contamination of the inanimate food processing environment is important and is highly likely to lead to the same organisms contaminating the foods being produced.6,7 The food industry in the 1980s and 1990s attracted the type of adverse media attention concerning cleanliness and risk management currently being received by hospitals. In response, the food industry put in place a range of corrective risk management strategies Professor Chris Griffith including Hazard Analysis Critical Control Points (HACCP), which could be combined with independent third party auditing in relation to standards and practices.8 While the food manufacturing industry is not perfect, hygiene standards and practices have improved as a result of the measures taken. Although not identical, there are interesting parallels in the survival, transfer and spread of pathogens in both food and healthcare environments. It has been argued in healthcare3 that it is the patients that contaminate the environment and not the reverse. If transmission one way is possible, it is difficult to believe the reverse cannot take place and studies have shown that reducing environmental contamination can lead to a reduction in infection rates9 and that contaminants from the environment can contaminate patients and ultimately lead to an infection.10 But the problem is complex and likely to involve many additional factors, including hand hygiene compliance rates, patient movement and bed occupancy levels. The potential for an environmental pathogen to contaminate foods is well recognised and has led to a much more organised and scientific approach to environmental monitoring.6 Environmental monitoring (see Table 1) is used in the food industry for plant commissioning, validation and bench marking of cleaning methods, as well as the more routine monitoring and verification of cleaning and is predicated on the belief that isolation of pathogens from the environment is a cause for concern. Another difference between the monitoring of cleaning in food and healthcare environments is how it is undertaken (Table 1). It is highly unlikely a major food processor would rely solely on visual measurement of cleanliness and would, for British Retail Consortium (BRC) certification, be required to have documented

Energy management The devil is in the details

The food industry believes the contamination of the inanimate food processing environment is likely to lead to the same organisms contaminating the foods being produced

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evidence of a monitoring programme and its results. Table 1: Comparison of approaches to monitoring cleaning efficacy between food and healthcare industries

FOOD ENVIRONMENT Isolation of pathogens from environmental surfaces causes concern Environmental surface sampling in food manufacturing used as part of a preventative strategy Range of surface sampling techniques used, including visual, microbiological and rapid methods in coordinated and integrated approaches

HEALTHCARE ENVIRONMENT Isolation of pathogens from environmental surfaces may or may not cause concern Environmental surface sampling likely to be used only in response to an outbreak Assessment of cleaning efficacy dominated by visual inspections (ICNA, PEAT, Healthcare Commission)

Monitoring cleanliness in the food industry starts with visual assessment (if visually dirty there is no point in other forms of measurement), but may then involve the measurement of residual soil using adenosine triphospahte (ATP). ATP is found in living cells and cell debris and is widely used in the food industry.6 While used by the top five world food manufacturing companies it has had only limited use in healthcare.11,12,13 These studies have shown clearly that visual assessment alone is an inaccurate measure of cleanliness and produces an verly optimistic indication of cleaning efficiency. Such findings should question the value of the development of elaborate methods of assessing cleanliness based solely on visual assessment.14 ATP provides a very rapid (10-20 seconds) measure of residual surface soil i.e. cleanliness, if cleaning is defined as the removal of soil. ATP can be used on its own and as a preliminary step prior to microbiological sampling. ATP and microbiological assessments measure different things (the latter measures only residual viable organisms). Although sometimes undertaken,15 there is therefore little value in trying to directly correlate one with another. For a strong correlation to exist, the ratios between organic debris and microorganisms would need to be constant and there are many reasons why this might not be the case.6 Of much greater benefit is comparing the results obtained using both approaches in relation to pass/fail limits following properly implemented cleaning.12,13 Surfaces passing ATP are much more likely to correlate with and achieve microbiological limits than those assessed visually (Table 2). Table 2: Surfaces passing visual assessment: Differences in failure rates after cleaning, between visual and two other assessment methods

ATP (% failure rates) ACC* (% failure rates) Hospital A Paediatric Surgical Hospital B Paediatric Surgical Hospital D Paediatric Surgical 77 82 93 83 77 91 45 51 62 84 73 77

*ACC= Aerobic Colony Count There is no ideal method for monitoring cleaning efficiency and in reality, as they measure different things ATP and microbiology should be used together as part of an integrated cost-effective assessment strategy.6 This considers not just the costs of testing but the costs of cleaning (which can be considerable and consider staff time, chemicals/equipment costs etc) as well as failure costs. Cleaning should deliver value for money (quality in relation to cost), and ATP can help to achieve this.6 However, not all ATP instruments are the same and some are more sensitive and reproducible than others and benchmark values obtained with one instrument/test are not applicable to values from other instruments/test combinations. Benchmark values after cleaning have been proposed and can be used in routine monitoring to improve the management of cleaning and in adopting a more scientific approach to decisions on cleaning frequency, as well as the validation of new cleaning methods. Evidence is growing that poor cleaning can result in increased environmental surface contamination and this may contribute to pathogen reservoirs16,17 and some cases of nosocomical infections caused by specific pathogens. An improved, more scientific assessment strategy may help to achieve better value for money from cleaning in healthcare as with the potential benefit of a reduction in infection rates caused by some pathogens. References 1. Boyce JM. J Hosp Infect 2007; 65 (Supp2): 50-54. doi: 10.10.1016/S0195-6701(07)60015-2 2. Dettenkofer M, Spencer RC. J Hosp Infect 2007; 65 (Supp2): 55-57. doi:10.1016/S0195-6701(07)60016-4 3. Fraise AP. J Hosp Infect 2007; 65 (Supp2): 58-59. doi:10.1016/S0195-6701(07)60017-6 4. Pratt RJ, Pellowe CM, Wilson JA, et al. J Hosp Infect 2007; 65 (supp 1): 1-29. doi: 10.1016S0195-6701(07)60002-4 5. Ayliffe GAJ, Fraise AP, Geddes AM, Mitchell K. Control of Hospital Infections: a

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practical Handbook. 4th edn. London: Hodder Arnold; 2002. 6. Griffth CJ. Improving surface sampling and detection of contamination, Handbook of Hygiene Control in the Food Industry. Cambridge: Woodhead Publishing; 2005. 7. Cordier JL. Assessing Microorganisms in Food and Factory. 3rd IAFP European Symposium on Food Safety. Rome: October 2007. 8. British Retail Consortium. Global Standard for Food Safety. Issue 5. TSO Stationary Office. 9. Denton M, Wilcox MH Parnell P, et al. J Hosp Infect 2004; 56: 106-110. 10. Hardy KJ, Oppenheim BA, Gossain S, Gao F, Hawkey PM. Infect Cont Hosp Epidemiol 2006; 27: 127-132. 11. Griffith, C.J., Cooper, R.A., Gilmore, J, Davies, C and Lewis M. (2000). Journal of Hospital Infection. 45(1): 19-28. 12. Griffith C.J., Obee P., Cooper R.A., Burton H.F., Lewis M (2007) Journal of Hospital Infection. June 66(4): pp352-359 13. Cooper R.A., Griffith C.J., Malik R.E., Obee P. and Looker N (2007) American Journal of Infection Control. 35 (5): pp 338-341 14. National Patient Safety Agency Report. The National Specifications for Cleanliness in the NHS: a framework for setting and measuring performance outcomes. NHS; April 2007. 15. Department of Health. Evaluation of ATP bioluminescence swabbing as a monitoring and training tool for effective hospital cleaning. Crown Copyright; 2007. 16. Sattar SA. Journal of Hospital Infection. 56, Supplement 2, April 2004: pp64-69 17. Sexton T, Clarke P, O'Neill E, Dillane T and Humphreys H (2006) Journal of Hospital Infection, 62(2): 133-260, February.

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Silliker, Inc., Food Science Center Report


RPN: 13922 December 11, 2009 Revised January 21, 2010

Performance Evaluation of Various ATP Detecting Units


Prepared for: Steven Nason Hygiena 941 Avenida Acaso Camarillo, CA 93012 Tel: 805-388-8007 snason@medicalpackaging.com Prepared by: Brian Kupski Operations Supervisor brian.kupski@silliker.com Erdogan Ceylan, Ph.D. Research Director erdogan.ceylan@silliker.com Cynthia Stewart, Ph.D. General Manager cynthia.stewart@silliker.com
The entire content of this REPORT is subject to copyright protection. Copyright 2009-10 Silliker, Inc. All rights reserved. The contents of this REPORT may not be copied other than for use by non-for-profit organization, and appropriate reference with all copyright notices stated. The REPORT may not be copied, reproduced or otherwise redistributed. Except as expressly provided above, copying, displaying, downloading, distributing, modifying, reproducing, republishing or retransmitting any information, text or documents contained in this REPORT or any portion thereof in any electronic medium or in hard copy, or creating any derivative work based on such documents, is prohibited without the express written consent of Silliker, Inc. Nothing contained herein shall be construed as conferring by implication, estoppel or otherwise any license or right under any copyright of Silliker, Inc., or any party affiliated with Silliker, Inc.

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Executive Summary
In early 2009 Silliker Group Corporation was commissioned to perform an extremely comprehensive study to examine the performance of several commercially available ATP (Adenosine Triphosphate) bioluminescence hygiene / sanitation monitoring systems. The properties of the ATP assay are well suited to determinations of cleanliness, with cleanliness being defined as the absence of organic (derived from life) material. Clean surfaces have little to no ATP, while dirty surfaces have ATP and perhaps live microbial cells. The result of an ATP test that is available in minutes permits the immediate assessment of the sample condition and whether additional cleaning action is required. The traditional method of determining cleanliness is the aerobic plate count. This test requires 2 days to complete. This procedure is limited in the type of microorganisms it can detect and does not detect organic residue. The study was designed to assess system performance when challenged with varying levels of pure ATP, food residues and microbes. There were 4 key study phases. Each phase of the study was designed to measure system performance under specific conditions against parameters chosen to approximate real world environmental situations and are vital technical measurements. It is important to understand that the combination of these factors and no single factor alone can clearly define system overall performance. Not all study phases include all systems. 1. Detection of pure ATP Swabs and solution 2. Detection of ATP from pure microbial cultures Escherichia coli, Lactobacillus plantarum, Pseudomonas aeruginosa, Salmonella Typhimurium and Staphylococcus aureus and one yeast culture- Saccharomyces cerevisiea 3. Detection of ATP from food Ground beef, Milk (pasteurized 2% low fat, Orange Juice (pasteurized without pulp),Salad (bagged mixed salad greens) 4. Detection of ATP from food soiled stainless steel surfaces Ground Beef, Milk At each stage of the study and for each combination of factors, 10 replicate sub-samples were tested at each test parameter. A total of >5000 data points were generated and these were analyzed mathematically to describe the performance of the tests in terms of Linearity, Repeatability, Sensitivity, and Accuracy. A key factor in the study of performance was the use of clone reagent swabs. These are reagent swabs that are formatted to operate with multiple instruments but are not proprietary to the instrument manufacturer. The study data indicates that the use of clone swabs is acceptable. Due to differences in RLU scales, data output (pass / fail settings based on RLU scale) and
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instrument linearity, the user must understand individual system nuances to successfully convert from a proprietary reagent swab to a clone. The key findings of the study are 1. 1. The linear correlation coefficient values for the ATP solution, microorganisms, and foods showed that the Log10 RLU readings and Log10 dilutions were linearly correlated. All Linear coefficient calculations can be found in tables 7 and 8 of the final report and were generally >0.9. However at low ATP levels , the Neogen and Charm systems lost linearity and could not detect <10 fmols ATP. 2. To quantify repeatability, the coefficient of variation (CV%) for each dilution of the ATP solution, microorganisms and foods were calculated. A high number of the average CV% values lies over the 10% to 35% range, which is reasonable for this type of assay and these types of studies. However the CV of individual systems for ATP detection ranged from 9% to 123%. The Hygiena SystemSURE with the Hygiena Supersnap swab had 9% CV whereas Neogen Accupoint and Charm Pocketswab had 123% and 86% CV respectively. 3. High RLU values do not confer a greater sensitivity to a system. Sensitivity depends greatly on several factors within the each system including both instrument and reagent output as well as the background of the system. The systems with large RLU scales such as Neogen and Charm systems also showed limit of detection of 10.0 fmols compared to other systems with a limit of detection of 1 fmol. 4. Clone swabs (Hygiena Snapshot) consistently improved the instrument performance by reducing the background noise and improving both sensitivity and repeatability of 3M CleanTrace, Charm Novalum and BioControl MVP systems. 5. The mean calculated sensitivity (or limit of detection) of the most and least sensitive systems differed by approximately 60 fold. The Hygiena SystemSure with the Hygiena Supersnap swab was the most sensitive reagent / instrument combination with a limit of detection of 0.17fmols. (data in tables 11 and 12) 6. When tested against the microbial cultures, the overall best extraction index included Hygiena Supersnap swab and Hygiena Snapshot swab. Several systems were included but no significant difference was observed.( tables 16, 17 and 18 ) The limit of detection was typically 10,000 to 100,000 bacteria / ml. 7. When tested against the food samples, the overall best extraction index included Hygiena Snapshot swab. Several systems were included but no significant difference in extraction rates was observed. ( tables 16, 17 and 18 ). However there was a difference in the sensitivity of the systems when testing foodstuffs. This was independent of the type of foodstuff and was similar to the sensitivities determined for ATP. The most sensitive system for the detection of food residues was Hygiena SystemSURE Plus with Supersnap swab and the least sensitive systems were the Neogen Accupoint and Charm Pocketswab. The report that follows this summary contains a large amount of detailed experimental data. It should be thoroughly reviewed to fully understand the depth of the experiments and the conclusions drawn from that data.

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Objective The purpose of this study was to evaluate commercial ATP units targeted for use in the food industry. The study was intended to provide comparative data and not as a comprehensive evaluation or review. Background ATP is a molecule that is essential and common to all plant, animal and microbial cells. ATP may persist long after the cells have died. Measurement of ATP requires only a few minutes and is based upon the firefly luciferase bioluminescence assay. The properties of the ATP assay are well suited to determinations of cleanliness, with cleanliness being defined as the absence of organic (derived from life) material. Clean surfaces have no ATP, while dirty surfaces have ATP and perhaps live microbial cells. The result of an ATP test that is available in minutes permits the immediate assessment of the sample condition and whether additional cleaning action is required. The traditional method of determining cleanliness is the aerobic plate count. This test requires 2 days to complete. This procedure is limited in the type of microorganisms it can detect and does not detect organic residue. ATP bioluminescence systems are available from a number of commercial companies. Measures of how these systems perform under controlled conditions will be helpful to customers as well as manufacturers that must make informed decisions. Materials and Methods ATP Monitoring Systems and Devices The study was conducted using two sets of ATP monitoring systems and swabs. In the first set (Set 1), the performance of five different commercially available ATP monitoring systems was evaluated using eight different commercially available swabs (Table 1). In the second set (Set 2), the performance of three different commercially available ATP monitoring systems of Set 1 was evaluated using four different commercially available swabs (Table 2). Table 1. First set of ATP monitoring system and swab combinations used in the sensitivity studies ATP monitoring system Swab Hygiena Snapshot SBC 1575 Biocontrol Lightning MVP Unit Lightning Clean Trace 3M Clean Trace NG Luminometer Hygiena Snapshot SPXL 1333 Hygiena Snapshot CH 1616 Charm Sciences novaLUM Unit Pocketswab Plus Hygiena SystemSURE Unit Hygiena Ultrasnap Neogen Accupoint Unit Neogen Accupoint

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Table 2. Second set of ATP monitoring system and swab combinations used in the sensitivity studies ATP monitoring system Swab Biocontrol Lightning MVP Unit Hygiena Snapshot SBC 1575 Clean Trace NG Luminometer Hygiena Snapshot SPXL 1333 Hygiena Supersnap Hygiena SystemSURE Unit Ultrasnap Test Matrices Test samples included water spiked with ATP as a measure of system sensitivity, microbial cultures and food soil representing organic residue likely to be present in environmental samples. The test samples, as described below, were prepared and analyzed by the ATP monitoring systems and swabs listed in Tables 1 and 2. Part A: Detection of Pure ATP The sensitivity of the ATP monitoring systems was first compared using water samples spiked at various levels of ATP (Table 3). In order to evaluate the influence of swab materials on ATP, the spiked water samples were tested either by depositing a predetermined amount of samples directly onto the swab bud or into the activated reagent in the swab device. For the blank (control) samples, a commercially available ultra pure sterile water product was used (Rockland Inc., Gilbertsville, PA). Table 3. ATP spiked water used in the sensitivity studies Recovery method ATP level (femtomole) ATP recovery from swab 0 0.1 1 5 10 100 1,000 ATP recovery in solution 0 100

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1. ATP recovery from swab a. 10 L of sample containing ATP levels at 0 (blank), 0.1, 1, 5, 10, 100, 1000 femtomoles (fmoles) was pipetted directly onto the appropriate swab bud. b. The swab device was activated. c. The swab was placed in the ATP unit. d. The measurement was started and the relative light units (RLU) result was read and recorded. e. This procedure was repeated with 10 replicates of each dilution on each swab device using each ATP system and swab combination of Set 1 1 and Set 2. f. Test results were reported as follows: ATP recovery from swab 1000 100 10 5 1 0.1 0 fmoles fmoles fmoles fmoles fmoles fmoles fmoles Replicates 1-10 2. ATP recovery in solution a. The swab device was activated without adding the sample. b. The swab was removed from the activated device. c. 10 L of sample containing ATP at the level of 0 (control blank) and 100 fmol was added into the reagent in the swab device. d. The swab was placed in the device. e. The swab device was placed in the ATP unit. f. The measurement was started and the RLU result was read and recorded. g. The ATP in solution tests were only performed on the following ATP system and swab combinations of Set1. i. Biocontrol Lightning MVP unit using Lightning swab, ii. Clean Trace NG Luminometer using Clean Trace swab, iii. Charm Science unit using Pocketswab Plus swab, iv. Hygiena SystemSURE unit using Hygiena Ultrasnap, v. Neogen Accupoint was omitted per clients request h. The ATP in solution tests were performed on all the ATP systems and swabs of Set 2. i. This procedure was repeated with 10 replicates of each dilution on each swab device using each ATP system and swab combination of Set 11 and Set 2. j. Test results were reported as follows: ATP recovery in solution 100 fmoles Replicates 1-10 0 fmoles (blank)

ATP systems and swabs of Set 1 were tested three times for each replicate, providing 30 readings
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Part B: Detection of ATP from pure microbial cultures grown in broth Five bacterial cultures, Escherichia coli, Lactobacillus plantarum, Pseudomonas aeruginosa, Salmonella Typhimurium and Staphylococcus aureus and one yeast cultureSaccharomyces cerevisiea were obtained from the Silliker Inc., Food Science Center culture collection (FSC-CC) (Table 4). The bacterial cultures were cultivated in 10 mL of tryptic soy broth (TSB) and incubated at 35C for 18-24 h. S. cerevisiea was cultivated in 10 mL of sabourand dextrose broth and incubated at 30C for 48 h. After incubation, each culture was washed once with sterile deionized water by centrifugation at 8000 rpm for 20 min and reconstitution with sterile deionized water. The cell level in each dilution of the bacterial cultures was determined by plating serial dilutions on tryptic soy agar (TSA) incubated 35C for 24 h. The cell level in each dilution of the yeast cultures was determined by plating serial dilutions on potato dextrose agar (PDA) incubated 25C for 5 d. Table 4. Bacterial strains used in the sensitivity studies conducted with pure cultures Culture Source FSC-CC Number Chicken broth 1809 Escherichia coli Juice drink 998 Lactobacillus plantarum Soil 2606 Pseudomonas aeruginosa ATCC MYA 658 2847 Saccharomyces cerevisiea Salmonella Typhimurium USDA culture 1860 Potatoes 1561 Staphylococcus aureus Each culture was serially diluted 10-fold up to 10-6 with sterile deionized water and analyzed by each ATP system and swab combination of Set 1 and Set 2 by pipetting 10 L of culture dilution directly onto each swab bud, placing the swab device into the ATP unit and reading the RLU result. After testing, the cell level in each dilution was determined by plating serial dilutions of the bacterial cultures on tryptic soy agar (TSA) and the yeast culture on potato dextrose agar PDA). The TSA plates were incubated at 35 1C for 24 2 h and the PDA plates were incubated at 30 1C for 48 h prior to enumeration. Test results were reported as follows: ATP from pure culture 0 dilution 1:10 1:100 1:1,000 1:10,000 1:100,000 1:1,000,000 (undiluted) dilution dilution dilution dilution dilution dilution Replicates 1-10 Part C: Detection of ATP from Food Food samples that are commonly available from commercial supermarkets were used to represent a range of product groups for this portion of the study (Table 5). Liquid food samples (orange juice and milk) were diluted using ATP-free sterile water (v/v) in the following ratios: full strength liquid (0 dilution); 1:10; 1:100; 1:1000; and 1:10,000. Solid food samples (ground beef and salad greens) were first stomached using 10 g of sample in 90 ml ATP-free sterile water and then diluted using ATP-free sterile water (w/w) in the following ratios: 1:10 (stomached samples); 1:100; 1:1,000; and 1:10,000. All test samples were shaken by hand for 2 min for homogenization.

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Table 5. Food samples used in the sensitivity studies Food Ground beef Milk (pasteurized 2% low-fat) Orange juice (pasteurized without pulp) Salad (bagged mixed salad greens) Ten replicates of each food suspension were analyzed by each ATP system and swab combination of Set 1 and Set 2 by pipetting 10 L of food suspension dilution directly onto each swab bud, placing the swab device into the ATP unit and reading the RLU result. Test results were reported as follows: ATP from Food 1:1 (full strength liquid; 1:10 1:100 0 dilution) Replicates 1-10 Part D: Detection of ATP from Food Soiled Stainless Steel Surfaces Some brands of swabs are wet, and stay wet, during the intended shelf life while other brands of swabs are dry. Test results may vary due to wet and dry swabs, and wet and dry surfaces. Therefore, in addition to the food suspension dilutions tested, stainless steel surfaces soiled with ground beef and milk were tested (Table 6). Table 6. Food suspension dilutions tested on stainless steel surface in the sensitivity studies. Food Dilution Ground beef 1:10 1:1,000 Milk 1:1 1:1,000 Five hundred (500) L of food suspension from the 1:10 and 1:1,000 dilutions of the ground beef and 500 L of food suspension from the 1:1 (full strength liquid; 0 dilution) and 1:1,000 dilutions of the milk and were spread evenly onto individual 4x4 in2 stainless steel surfaces and immediately tested after preparation by swabbing each swab bud over the stainless steel surface, placing the swab device in the ATP unit and reading the RLU result. An additional set of stainless steel surfaces were prepared as described above and allowed to dry at room temperature for 18-24 h. After drying, each individual stainless steel piece was swabbed by each swab bud and analyzed by each ATP unit and reading the RLU result. Ten replicates of each dilution were tested. Test results were reported as follows: ATP from Food Soiled Stainless Steel Surface 1:1 (full strength 1:10 1:1,000 liquid; 0 dilution) Replicates 1-10 1:1,000 1:10,000

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Data Analysis Linearity: The linear correlation coefficient (r) measures the strength and the direction of a linear relationship between the sample variables. The value of r for samples at varying ATP levels and food samples at varying dilution levels against the corresponding RLU readings was calculated to determine the linear relationship. Repeatability Repeatability means the level of agreement between successive results obtained with the same method on the same test sample. The ATP measurement repeatability was expressed as a coefficient of variation (CV%), which is the standard deviation (SD) expressed as percentage of the mean (i.e. CV % = 100 SD/mean). Sensitivity Limit of detection (LOD) in this study was calculated as the ratio of the mean of a true blank with three standard deviations to RLU per fmoles (mean +3 sd/RLU per fmoles). Some systems such as the SystemSure, the blanks can run at 0, the next significant RLU is used as the lowest detection limit. Charm and Neogen instruments have a built in algorithm, which discounts some of the RLU measured and therefore they do not display an RLU value for less than 10 fmoles. For these systems an LOD value of 10 fmoles is stated. Relative Light Unit (RLU) per femtomole The RLU per femtomole values were calculated by dividing RLU readings to corresponding ATP levels. In order to minimize the variability, the average RLU per femtomole was calculated using the first three ATP dilutions (i.e. 1,000, 100 and 10 fmoles). Comparison of index (extraction index) for microorganisms and food samples The lowest detectable concentration levels for microbial and food samples are presented as the lowest dilution at which ATP could be detected. The extracted fmole per dilution was calculated as the ratio of the RLU reading without background to the RLU per fmole value. The extracted fmole per dilution values that show greater than 1 ATP fmole are counted as extracted. The comparison using the extracted ATP rather than RLU normalizes the data making analysis comparable and extraction levels more relevant to each system.

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Results and Discussion Test results are reported in relative light unit (RLU) readings in this study. The sensitivity of each of the 12 ATP monitoring systems of Set 1 and Set 2 was tested using aqueous ATP solutions, cell suspensions and exudates of food samples. Each ATP monitoring system uses a different measurement scale. The test results of ten replicates of seven dilutions of the ATP solution, seven dilutions of cell suspensions, five dilutions of liquid food samples (i.e. milk and orange juice), four dilutions of solid food samples (ground beef and salad) and two dilutions of food exudates on stainless steel surfaces are shown in Appendix A. The formulation Hygiena swab products used in Set 1 and Set 2 differ in order to show the effects of extractant on subsequent detection and test performance. Hygiena products used in Set 2 are those supplied routinely on a commercially basis. Linearity Log10 RLU values of ten replicates were plotted against Log10 dilutions of the test matrices (Appendix B). The best-fit line is represented by the solid line and the 95% confidence limits presented as dashed lines. The linear correlation coefficient (r) of the best-fit line was determined to measure the linearity of ATP monitoring systems. A value of 1.0 represents a perfect fit of the regression line to the data. Values greater than 0.8 indicate the curve fits the data very well. The linear correlation coefficient values of the ATP monitoring systems tested for RLU over the range of dilutions of the ATP solution, microorganisms, and foods are summarized in Tables 7 and 8. All regressions were significant. A total of 132 correlation coefficients were calculated. All correlation coefficient values with the exception of an outlier value of 0.643 determined by the Neogen Accupoint with Neogen Accupoint swab for E. coli, were greater than 0.8 and provided strong evidence that the Log10 RLU readings and Log10 dilutions were linearly correlated. Repeatability To quantify repeatability, the coefficient of variation (CV%) for each dilution of the ATP solution, microorganisms and foods were calculated (Appendix C). The CV% values indicate the amount of variation. The higher CV% values represent greater variation and hence less repeatability. The CV% values increased as the limits of detection were approached. This is expected because closer to the detection limit there is much less ATP to measure and there is more variability in the measurement. The CV% were erratic between dilutions of the test matrices and ranged from 2% to 316%. For comparative purposes, the average CV% of the ATP solution data, microbial cultures and food samples was calculated. All CV% were then averaged for each ATP monitoring system (Table 9-10). The average coefficient of variation values ranged from a low of 6% by the BioControl Lightning MVP with Hygiena Snapshot SBC 1575 swab to a high of 186% by the Neogen Accupoint with Neogen Accupoint swab. The relative frequency distribution of the average CV% values is presented in Figure 1. A high number of the average CV% values lies over the 10% to 35% range, which is reasonable for this type of assay and these types of studies. The natural variation of biological assays such as ATP bioluminescence combined with the variation from sample collection and operator handling during hygiene monitoring applications means that the test results do not have the same precision as other analytical methods. The results from ATP hygiene measurements are used as a rapid qualitative Copyright 2009-10 Silliker, Inc. 10

assessment of cleaning and results are typically expressed in board bands of Pass , Caution or Fail that typically equate to 10 100fmols of ATP. The ATP hygiene monitoring application is not intended to be used as a precise determination of ATP content. The trending of RLU or Pass / Fail results are much more meaningful in routine manufacturing operations. Sensitivity ATP Solution The average RLU readings, standard deviations and CV% values for dilution of the ATP solution, microorganisms and food samples are summarized in Appendix C. The average background readings from the system (i.e. reagents outputs in the 0.0% ATP blank solution) for the 10 replicate test samples were 0.0 RLU for the swab devices of Charm Science with Pocketswab Plus swab (Set 1), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1) (Appendix C-Table C1) and the Hygiena SystemSure with Hygiena Supersnap swab (Set 2) and Hygiena SystemSure with Hygiena Ultrasnap swab (Set 2) (Appendix C Table C2). The average background reading for the Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575 swab, BioControl Lightning MVP with Lightning swab, Clean Trace NG Luminometer with Clean Trace swab, Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab, and Hygiena SystemSure with Hygiena Ultrasnap swab of Set 1 were 142.17, 283.17, 4.0, 0.83, and 0.67 RLU, respectively (Appendix C-Table C1). The average background reading for the Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575swab and Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab were 199 and 1.90 and RLU, respectively (Appendix C-Table C2). ATP analysis of the ATP solutions showed that the calculated LODs ranged from 0.17 fmoles to 10 fmoles (Tables 11 and 12). The mean calculated LODs of the most and least sensitive systems differed by approximately 60 fold. The Hygiena SystemSure with Hygiena Supersnap swab was the most sensitive, while the Charm Science with Pocketswab Plus swab and the Neogen Accupoint with Neogen Accupoint swab were the least sensitive systems. The RLU output and range shown of different systems varies considerably because the RLU is not a standard unit of measurement and is unique to each test system. High RLU values do not confer a greater sensitivity to a system and this is shown in Table 13 that summarizes the performance characteristics of the tests systems as supplied commercially. ATP detection and recovery was variable between systems (Figure 2). BioControl had a high recovery of ATP but it was also highly variable (+/- 37%). Hygiena ATP recovery was high (92%) with good repeatability (9% CV). The Charm Science with Pocketswab recovered only 57% ATP with 20% variability, and Clean Trace NG Luminometer systems recovered only 52% ATP with a variability of 10%. A reduced recovery of ATP means that the accuracy of the system is also reduced. Hygiena SnapShot is designed to be used with other luminometers such as 3M Clean Trace NG Luminometer, BioControl MVP and Charm Sciences novaLUM. Table 14 shows snapshot performance for ATP detection compared to other systems and their corresponding swabs.

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Snapshot increases the performance of other luminometers and detects lower levels of ATP by; Increasing the linearity of ATP response Reducing the background and giving similar or greater RLU output per unit of ATP Reducing the variation and thereby increasing repeatability and consistency of ATP detection Increasing the extractability of ATP and thereby increasing the accuracy of the measurement Improving the sensitivity of the system Similar results were also obtained with the detection of foodstuffs. Microorganisms Data for six different microbial cultures using various ATP systems and swab devices are presented in Appendix C-Table C3 through C14. During the course of testing the dilutions, the lower detection limit was observed; hence not all 10-fold dilutions were analyzed by each swab device. The analysis of each culture used the RLU per femtomole calculation to normalize the RLU measured by each system to femtomoles. This normalization is required to bring all measured RLUs onto a similar scale; this scale can then be easily compared device to device and instrument to instrument. Comparisons using RLUs is difficult due to the differing scales used and the variable machine and reagent background RLUs which do not contribute to the measured signal. Hence, the normalization of the data to RLU per femtomole is required for accurate comparative. Escherichia coli E. coli had a culture level of 9.59 log10 CFU/ml for the first set of swab devices (Set 1) analyzed and 9.08 log10 CFU/ml for the second set of swab devices (Set 2) tested. The Biocontrol Lightning MVP with Hygiena Snapshot 1575 swab (Set 1), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1), Hygiena SystemSure with Hygiena Ultrasnap swab (Set 1 and 2), and Neogen Accupoint with Neogen Accupoint swab (Set 1) were the least sensitive swab devices analyzed for the detection of ATP from E. coli as these swab devices were able to detect the E. coli at the 1:100 diluted culture level, and not when the culture was subsequently diluted (Appendix C-Table C3, Appendix D Table D2). The Biocontrol Lightning MVP with Lightning swab (Set 1), Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 2), Clean Trace NG Luminometer with Cleantrace swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot 1333 swab (Set 1 and 2), Charm Science with Pocketswab (Set 1) and Hygiena SystemSure with Hygiena Supersnap (Set 2) appeared to be the most sensitive swab devices analyzed as these were able to detect ATP from E. coli at the next dilution (1:1,000) tested (Appendix C-Tables C3 and C4, Appendix D Table D2). Lactobacillus plantarum L. plantarum had a culture level of 6.45 log10 CFU/ml for the first set of swab devices (Set 1) analyzed and 9.48 log10 CFU/ml for the second set of swab devices (Set 2) tested. The Biocontrol Lightning MVP with Lightning swab (Set 1), Hygiena SystemSure with Hygiena Ultrasnap swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1) were the least sensitive swab devices analyzed for the detection of ATP from L. plantarum, as these swab devices were able to detect L. plantarum at the 1:10 diluted culture level (Appendix C-Table C5, Appendix D Table D2). The Biocontrol MVP with Hygiena Snapshot 1575 swab (Set 1), Clean Trace NG Luminometer with Cleantrace
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swabs (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot 1333 swab (Set 1), Charm Science with Hygiena Snapshot 1616N swab (Set 1), Charm Science with Pocketswab (Set 1), and Hygiena SystemSure with Hygiena UltraSnap swab (Set 2) could extract ATP from the next dilution (1:100). The most sensitive swabs were Biocontrol Lightning MVP with Hygiena Snapshot 1575 swab (Set 2), Clean Trace NG Luminometer with Hygiena Snapshot 1333 swab (Set 2) and Hygiena SystemSure with Hygiena Supersnap swab (Set 2), as these were able to detect ATP from L. plantarum at the 1:1,000 dilution level. (Appendix C-Tables C5 and C6, Appendix D Table D2). Pseudomonas aeruginosa P. aeruginosa had a culture level of 8.32 log10 CFU/ml for the first set of swab devices (Set 1) analyzed and 8.52 log10 CFU/ml for the second set of swab devices (Set 2) tested. The Clean Trace NG Luminometer with Cleantrace swab (Set 1), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1), Charm Science with Pocketswab Plus swab (Set 1), Hygiena SystemSure with Hygiena Ultrasnap swab (Set 1 and 2), Neogen Accupoint with Neogen Accupoint swab (Set 1) and Hygiena SystemSure with Hygiena Supersnap swab (Set 2) were the least sensitive swab devices analyzed for the detection ATP from P. aeruginosa, as these swab devices were able to detect P. aeruginosa at the 1:100 diluted culture level, and not when the culture was subsequently diluted (Appendix C-Tables C7 and C8, Appendix D Table D2). The Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 1 and Set 2), Biocontrol Lightning MVP with Lightning swab (Set 1) and Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 1 and 2) appeared to be the most sensitive swab devices analyzed as these were able to detect ATP from P. aeruginosa at the 1:1,000 diluted culture level (Appendix C-Tables C7 and C8, Appendix D Table D2). Saccharomyces cerevisiae S. cerevisiae had a culture level of 7.49 log10 CFU/ml for the first set of swab devices (Set 1) analyzed and 7.74 log10 CFU/ml for the second set of swab devices (Set 2) tested. The Charm Science with Hygiena Snapshot CH 1616 swab (Set 1), Charm Science with Pocketswab Plus swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1) were the least sensitive swab devices analyzed for the detection of ATP from S. cerevisiae as these swab devices only were able to detect S. cerevisiae at the 1:1,000 diluted culture level, while all other systems could detect ATP from S. cerevisiae at the 1:10,000 diluted culture level (Appendix C-Tables C9 and C10, Appendix D Table D2). Salmonella Typhimurium S. Typhimurium had a culture level of 7.96 log10 CFU/ml for the first set of swab devices (Set 1) analyzed and 9.08 log10 CFU/ml for the second set of swab devices (Set 2) tested. The Biocontrol Lightning MVP with Hygiena Snapshot 1575 swab (Set 1), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1) were the least sensitive swab devices analyzed for the detection of ATP from S. Typhimurium as these swab devices were able to detect ATP from S. Typhimurium at the 1:10 diluted culture level, while the Biocontrol Lightning MVP with Lightning swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 1), Charm Science with Pocketswab (Set 1) and Hygiena SystemSure with Hygiena Ultrasnap swab (Set 1 and 2) could detect ATP from S. Typhimurium at the 1:100 diluted culture level. The most sensitive systems were Clean Trace NG Luminometer with Cleantrace swab (Set 1), Biocontrol Lightning MVP with Hygiena Snapshot 1575 swab (Set 2), Clean Trace NG Luminometer with Hygiena
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Snapshot SPXL 1333 swab (Set 2) and Hygiena SystemSure with Hygiena Supersnap swab (Set 2) as these swab devices were able to detect ATP from S. Typhimurium at the 1:1,000 diluted culture level (Appendix C-Tables C11 and C12, Appendix D Table D2). Staphylococcus aureus S. aureus had a culture level of 8.23 log10 CFU/ml for the first set of swab devices (Set 1) analyzed and 8.76 log10 CFU/ml for the second set of swab devices (Set 2) tested. The Biocontrol Lightning MVP with Snapshot 1575 swab (Set 1), Biocontrol Lightning MVP with Lightning swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 1), Charm Science with Pocketswab (Set 1), Hygiena SystemSure with Hygiena UltraSnap swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1) were the least sensitive swab devices analyzed for the detection of ATP from S. aureus, as these swab devices were able to detect ATP from S. aureus at the pure culture level, and no detection was observed at any lower levels of S. aureus (Appendix C-Table C13, Appendix D Table D2). The Clean Trace NG Luminometer with Cleantrace swab (Set 1) and Hygiena SystemSure with Hygiena UltraSnap swab (Set 2) could detect ATP from S. aureus at the 1:10 diluted culture level, while Charm Science with Hygiena Snapshot CH 1616 swab (Set 1), Biocontrol Lightning MVP with Hygiena Snapshot 1575 swab (Set 2), Clean Trace NG Luminometer with Hygiena Snapshot 1333 swab (Set 2) were able to detect ATP from S. aureus at the 1:100 diluted culture level. The most sensitive swab device analyzed for the detection of ATP from S. aureus was the Hygiena SystemSure with Hygiena Supersnap swab (Set 2) as it was able to detect ATP from S. aureus at the 1:1,000 diluted culture level (Appendix C-Tables C13 and C14, Appendix D Table D2). Compendium Extraction Index To fully evaluate how the systems perform across the range of bacteria, the lowest level from each system for each bacterium can be assessed by analyzing at which dilution level in each dilution series 1 femtomole of ATP can be extracted above the blank values. This relationship is then tabulated in Table 15. The systems with the overall best extraction index include Hygiena SystemSURE with Hygiena Supersnap swab (Set 2), Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 2) and Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 2). The overall extraction index is -3.00 (which is a mean extraction level of 1:1,000) across all bacteria measured. The next best systems are in the -3.00 to -2.00 range (i.e. 1:100 to 1:1000 dilution region). These systems include Clean Trace NG Luminometer with Clean Trace swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 1), Hygiena SystemSURE with Hygiena Ultrasnap swab (Set 2), Biocontrol Lightning MVP with Lightning swab (Set 1), Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 1), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1) and Charm Science with Pocketswab Plus swab (Set 1). With the other systems with extractions below the 1:100 dilution across all bacteria includes Hygiena SystemSURE with Hygiena Ultrasnap swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1). Large differences were observed in the ATP results from different species of microorganism and these were lower than expected. This may be a reflection of species difference and size or the effect of the culture preparation and sample storage during testing. The limit of detection for most systems was 105 106 bacteria CFU/ml which equates to 103 104 bacterial per swab. Similarly 103 104 yeast/ml which equates to 101
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102 yeasts per swab. However the prime purpose of the ATP hygiene monitoring application is to detect product residue after cleaning because residues are a direct objective measurement of cleaning efficiency, and the level of ATP in foodstuffs is far greater than that of microbes. The ATP test is not intended to be a replacement for microbiological tests. The post-cleaning standard for bacteria on surfaces is typically 100 500 CFU per 100 cm2 which is equivalent to 100 500 CFU per swab that is clearly not detectable by the ATP test as shown above. Food Samples Raw Ground Beef The Biocontrol Lightning MVP with Lightning swab (Set 1) was the least sensitive swab device analyzed for the detection of ATP for raw ground beef as this swab device only was able to detect this food suspension at the 1:10 dilution level, while Biocontrol Lightning with Hygiena Snapshot 1575swab (Set 1), Clean Trace NG Luminometer with Cleantrace swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshots 1333 swab (Set 1 and Set 2), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1), Charm Science with Pocketswab Plus swab (Set 1), and Neogen Accupoint with Neogen Accupoint swab (Set 1) could detect ATP at the 1:100 dilution level. Hygiena SystemSure with Hygiena Ultrasnap swab (Set 1 and Set 2) and Hygiena SystemSure with Hygiena Supersnap swab (Set 2) were able to detect ATP for raw ground beef at the 1:1,000 dilution level. The most sensitive swab device was BioControl Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 2) as this swab device was able to detect ATP from raw ground beef at the lowest (1:10,000) dilution tested (Appendix C-Tables C15 and C16, Appendix D Table D1). Milk The Neogen Accupoint with Neogen Accupoint swab (Set 1) was the least sensitive swab device analyzed for the detection of ATP from pasteurized 2% low fat milk as this swab device only was able to detect this food suspension at the 1:10 dilution level, while the BioControl Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 1), BioControl Lightning MVP with Lightning swab (Set 1), Clean Trace NG Luminometer with Clean Trace swab (Set 1), Charm Science with Hygiena Snapshot CH 1616 swab (Set 1), Charm Science with Pocketswab Plus swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 2) and Hygiena SystemSURE with Hygiena Supersnap swab (Set 2) could detect ATP from this food suspension at the 1:100 dilution level. Hygiena SystemSure with Ultrasnap swab (Set 1) and BioControl Lightning MVP with Hygiena Snapshot 1575 swab (Set 2) were able to detect ATP at the 1:1,000 dilution level. The most sensitive swabs were Clean Trace NG Luminometer with Hygiena Snapshot 1333 swab (Set 1) and Hygiena SystemSure with Hygiena Ultrasnap swab (Set 2) as these devices were able to detect ATP from pasteurized 2% low fat milk at the 1:10,000 dilution level (Appendix C-Tables C17 and C18, Appendix D Table D1). Orange Juice The Charm Science with Pocketswab Plus swab (Set 1), Hygiena SystemSure with Ultrasnap swab (Set 1) and Neogen Accupoint with Neogen Accupoint swab (Set 1) were the least sensitive swab devices analyzed for the detection of ATP from orange juice containing no pulp as these swab devices only were able to detect this food suspension at the 1:1,000 dilution level, while all other systems analyzed were able to detect ATP for orange juice at the lowest dilution (1:10,000) tested (Appendix C-Tables C19 and C20, Appendix D Table D1).
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Mixed Salad Greens The Charm Science with Hygiena Snapshot CH 1616 swab (Set 1) was the least sensitive swab device analyzed for the detection of ATP from orange as this swab device only was able to detect this food suspension at the 1:100 dilution level, while Clean Trace NG Luminometer with Cleantrace swab (Set 1), Charm Science with Pocketswab Plus swab (Set 1), Hygiena SystemSure with Hygiena Ultrasnap swab (Set 1), Neogen Accupoint with Neogen Accupoint swab (Set 1), and Clean Trace NG Luminometer with Snapshot 1333 (Set 2) could ATP from bagged mixed salad greens at the 1:1,000 dilution level (Appendix C-Table C21, Appendix D Table D1). The most sensitive swab devices analyzed for the detection of ATP for bagged mixed salad greens were the BioControl Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 1), BioControl Lightning MVP with Lightning swab (Set 1), Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 1), Biocontrol Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 2), Hygiena SystemSURE with Hygiena Supersnap swab (Set 2) and Hygiena SystemSURE with Hygiena Ultrasnap swab (Set 2) as these were able to detect ATP from salad greens at the lowest dilution (1:10,000) tested (Appendix C-Tables C21 and C22, Appendix D Table D1). Wet versus Dry Food Soil All swab devices analyzed were able to detect ATP from the wet and dry soiled stainless steel surfaces from the different food suspensions of raw ground beef and pasteurized 2% low fat milk (Appendix C-Tables C23-C26). The RLU reading of the wet and dry soiled stainless steel surfaces were higher than that of food suspension at the same dilution levels. This may be attributed to the difference in volumes used for food suspensions (i.e. 10 L) and stainless steel coupons (i.e. 500 L). Snapshot improved sample recovery from dry surfaces (Figure 3). This is attributed to snapshots saturated swab bud and extractant that ensures good recovery of sample and increase RLU output compared to suppliers own swab. Overall Comparison For overall comparative purposes, the average extraction index of each microbial and food sample, and the ATP monitoring system were calculated. When tested against the microbial cultures, the Hygiena SystemSure system with Hygiena Supersnap swab (Set 2), BioControl Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 2) and Clean Trace NG Luminometer with Hygiena Snapshot SPXL 1333 swab (Set 2) appeared to be the most sensitive ATP monitoring system analyzed as they were able to detect ATP from the microbial cultures at higher dilution levels compare to all other systems (Table 15). Most RLU output due to ATP derived from microbial cultures was highest in S. cerevisiae and lowest in S. aureus. When tested against the food samples, the Hygiena SystemSure with Hygiena Ultrasnap swab (Set 2) and the BioControl Lightning MVP with Hygiena Snapshot SBC 1575 swab (Set 2) appeared to be the most sensitive ATP monitoring systems analyzed as they were able to detect ATP from the food samples at higher dilution levels compare to all other systems (Table 16). Most RLU output due to ATP derived from food residues was highest in orange juice and lowest in ground beef. Each system with generic or clone swabs can be tabulated and graded according to performance from each section, aqueous ATP detection, ATP recovery from swab, extraction of ATP from microbial cultures and extraction of ATP from food stuffs. The
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comparison is shown in Table 17, each category ranks the systems using the most current versions of each system either swab or instrument. The Hygiena swabs collectively are more sensitive to ATP and better at detecting low level food and cultures than all other systems.

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Table 7. Correlation coefficient of ATP monitoring systems of Set 1


Microorganism Food Salmonella Typhimurium 0.947 Staphylococcus aureus 0.868 Ground beef 0.951 Milk Orange juice 0.981 Salad

ATP Unit

Swab Device Hygiena Snapshot SBC 1575 Lightning Clean Trace Hygiena Snapshot SPXL 1333 Hygiena Snapshot CH 1616 Pocketswab Plus Hygiena Ultrasnap Neogen Accupoint

ATP Solution 0.987

Escherichia coli 0.986

Lactobacillus plantarum 0.955

Pseudomonas aeruginosa 0.995

Saccharomyces cerevisiea 0.984

Biocontrol Lightning MVP Biocontrol Lightning MVP Clean Trace NG Luminometer Clean Trace NG Luminometer Charm Science Charm Science Hygiena SystemSURE Neogen Accupoint

0.972

0.998

0.982

0.931

0.928

0.986

0.989

0.921

0.810

0.945

0.960

0.996

0.992

0.988

0.993

0.975

0.989

0.988

0.974

0.920

0.890

0.974

0.997

0.995

0.988

0.987

0.985

0.992

0.997

0.990

0.984

0.855

0.986

0.984

0.996

0.982 0.949 0.988 0.976

0.984 0.972 0.962 0.643

0.998 0.997 0.991 0.970

0.995 0.984 0.974 0.972

0.984 0.983 0.979 0.928

0.945 0.983 0.980 0.967

0.997 0.998 ND a 0.961

0.954 0.990 0.855 0.995

0.909 0.982 0.986 0.920

0.971 0.986 0.984 0.938

0.937 0.986 0.996 0.985

Not determined; only 0 dilution had RLU readings.

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Table 8. Correlation coefficient of ATP monitoring systems of Set 2


Microorganism Food Salmonella Typhimurium 0.997 Staphylococcus aureus 0.986 Ground beef 0.964 Milk Orange juice 0.991 Salad

ATP Unit

Swab Device Hygiena Snapshot SBC 1575 Hygiena Snapshot SPXL 1333 Hygiena Supersnap Hygiena Ultrasnap

ATP Solution 0.990

Escherichia coli 0.999

Lactobacillus plantarum 0.986

Pseudomonas aeruginosa 0.989

Saccharomyces cerevisiea 0.995

Biocontrol Lightning MVP Clean Trace NG Luminometer Hygiena SystemSURE Hygiena SystemSURE

0.976

0.933

0.992

0.998

0.978

0.991

0.982

0.997

0.952

0.978

0.988

0.993

0.997

0.987 0.989

0.987 0.974

0.990 0.989

0.991 0.991

0.980 0.991

0.996 0.989

0.992 0.968

0.984 0.966

0.986 0.985

0.950 0.978

0.986 0.985

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Table 9. Coefficient of variation (CV%) of ATP solution, microorganisms and food samples when tested by different ATP monitoring systems-Set 1
Swab Device Hygiena Snapshot SBC 1575 Lightning Clean Trace Hygiena Snapshot SPXL 1333 Hygiena Snapshot CH 1616 Pocketswab Plus Hygiena Ultrasnap Neogen Accupoint Escherichia coli 20 62 24 22 7 45 28 186 Lactobacillus plantarum 18 22 20 16 8 14 15 30 Pseudomonas aeruginosa 24 51 24 31 16 31 51 36 Saccharomyces cerevisiea 53 31 27 25 16 30 31 63 Salmonella Typhimurium 20 49 27 20 18 15 20 142 Staphylococcus aureus 20 30 23 24 14 15 11 113 Ground beef 19 15 15 22 15 22 31 78 Orange juice 14 20 15 17 9 17 17 19 Ground beef soiled surface 35 24 31 30 26 19 58 33 Milk soiled surface 15 32 23 29 15 22 50 27

ATP Unit Biocontrol Lightning MVP Biocontrol Lightning MVP Clean Trace NG Luminometer Clean Trace NG Luminometer Charm Science Charm Science Hygiena SystemSURE Neogen Accupoint

APT Solution 19 39 26 27 68 86 59 123

Milk

Salad

Average (Range) 2 22 (12-53) 34 (15-62) 22 (10-31) 23 (10-31) 19 (7-68) 28 (14-86) 33 (11-59) 68 (17-186)

21 52 18 26 22 31 40 19

12 20 10 10 19 17 17 17

CV% Average does not include 0 and NA readings

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Table 10. Coefficient of variation of ATP solution when tested by different ATP monitoring systems-Set 2
Swab Device Hygiena Snapshot SBC 1575 Hygiena Snapshot SPXL 1333 Hygiena Supersnap Hygiena Ultrasnap Escherichia coli Lactobacillus plantarum Pseudomonas aeruginosa Saccharomyces cerevisiea Salmonella Typhimurium Staphylococcus aureus Ground beef Orange juice Ground beef soiled surface Milk soiled surface

ATP Unit Biocontrol Lightning MVP Clean Trace NG Luminometer Hygiena SystemSURE Hygiena SystemSURE

APT Solution

Milk

Salad

Average (Range) 3

10

11

10

44

28

19

17

48

16

19

21

25

21 (6-48) 23 (13-44) 23 (9-41) 30 (12158)

15 9 28

18 18 15

14 17 13

35 41 30

17 14 18

20 17 12

16 18 14

27 34 13

19 32 158

13 18 13

18 18 17

36 28 23

44 34 32

CV% Average does not include 0 and NA readings

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Figure 1. Relative frequency distribution for CV% values

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Relative Frequency (Percentage) Distribution

25

20

15

10

0 >5 6-10 11-15 16-20 21-25 26-30 31-35 CV% Range APT Solution Microbial Culture Food Soil 36-40 41-45 46-50 51-55 56-60 > 60

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Table 11. Relative Light Unit (RLU) per femtomole (fmole) and limit of detection (LOD) values of Set 1 ATP Unit Biocontrol Lightning MVP Biocontrol Lightning MVP Clean Trace NG Luminometer Clean Trace NG Luminometer Charm Science Charm Science Hygiena SystemSURE Neogen Accupoint Swab Device Hygiena Snapshot SBC 1575 Lightning Clean Trace Hygiena Snapshot SPXL 1333 Hygiena Snapshot CH 1616 Pocketswab Plus Hygiena Ultrasnap Neogen Accupoint RLU/fmole 552 698 5.4 6.8 582 218 1.0 12 LOD (fmole) 0.60 1.10 1.30 0.42 5.0 10.0 1.0 10.0

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Table 12. Relative Light Unit (RLU) per femtomole (fmole) and limit of detection (LOD) values of Set 2 ATP Unit Biocontrol Lightning MVP Clean Trace NG Luminometer Hygiena SystemSURE Hygiena SystemSURE Swab Device Hygiena Snapshot SBC 1575 Hygiena Snapshot SPXL 1333 Hygiena Supersnap Hygiena Ultrasnap RLU/fmole 825 9.0 6 1.0 LOD (fmole) 0.40 0.39 0.17 1.0

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Table 13: Summary of ATP performance characteristics of 5 commercial detection systems System Linearity (r) BioControl MVP with Lightning swab 3M Clean Trace NG Luminometer with CleanTrace swab Charm Science novaLUM with Pocketswab Plus Hygiena SystemSURE Plus with Ultrasnap swab Hygiena SystemSURE Plus with Supersnap swab Neogen AccuPoint With Accupoint swab 0.982 0.988 0.949 0.988 0.987 0.976 Output (RLU) Blank (Background at zero ATP) 283 4 0 0 0 0 Maximum (at 1000 fmols ATP ) 975,941 7382 418,517 * 1589 4949 15,649 * Variability (CV%) 39 26 86 28 9 123 Sensitivity Limit of detection (fmols ATP) 1.1 1.3 10.0 1.0 0.17 10.0

* does not detect below 10 fmols at which level the instrument shows 0 RLU.

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Figure 2: Recovery of ATP by different ATP detection systems


160 140 120 100 ATP recovery (%) 80 60 40 20 0 Hygiena SystemSURE BioControl MVP Charm Sciences novaLUM 3M Clean Trace NG Luminometer

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Table 14: SnapShot performance in different luminometer compared to manufacturers own swabs System Linearity (r) BioControl MVP with Lightning swab BioControl MVP with Snapshot swab 3M Clean Trace NG Luminometer with CleanTrace swab 3M Clean Trace NG Luminometer with Snapshot swab Charm Science novaLUM With Pocketswab Plus Charm Science novaLUM with Snapshot swab 0.982 0.990 0.988 0.992 0.949 0.982 Output (RLU) Blank (Backgrounda t zero ATP) 283 199 4 2 0 0 Maximum (at 1000 fmols ATP ) 975,941 927,161 7382 12620 418,517 783,031 Variability (CV%) 39 10 26 15 86 68 Sensitivity Limit of detection (fmols ATP) 1.1 0.4 1.3 0.42 10.0 5.0

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Figure 3: Snapshot sample recovery from dry surfaces compared with other swabs

4500 4000 3500 RLU output 3000 2500 2000 1500 1000 500 0 BioControl Meat residue 3M Clean Trace NG Meat residue Suppliers swab Hygiena snapshot 3M Clean Trace NG Milk residue

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Table 15. Summary of lowest dilution levels for RLU output due to ATP derived from microbiological culture dilutions tested by different ATP monitoring systems

ATP Unit Hygiena SystemSURE (Set 2) Biocontrol Lightening MVP (Set 2) Clean Trace NG Luminometer (Set 2) Clean Trace NG Luminometer (Set 1) Clean Trace NG Luminometer (Set 1) Hygiena SystemSURE (Set 2) Biocontrol Lightening MVP (Set 1) Biocontrol Lightening MVP (Set 1) Charm Science (Set 1) Charm Science (Set 1) Hygiena SystemSURE (Set 1) Neogen Accupoint (Set 1)

Swab Device Hygiena Supersnap Hygiena Snapshot SBC 1575 Hygiena Snapshot SPXL 1333 Clean Trace Hygiena Snapshot SPXL 1333 Hygiena Ultrasnap Lightening Hygiena Snapshot SBC 1575 Hygiena Snapshot CH 1616 Pocketswab Plus Hygiena Ultrasnap Neogen Accupoint

Order of best extraction (mean of lowest dilutions detected) -3.00 -3.00 -3.00 -2.50 -2.33 -2.17 -2.17 -2.00 -2.00 -2.00 -1.83 -1.50

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Table 16. Summary of lowest dilution levels for RLU output due to ATP derived from food samples suspensions tested by different ATP monitoring systems ATP Unit Hygiena SystemSURE (Set 2) Biocontrol Lightening MVP (Set 2) Clean Trace NG Luminometer (Set 1) Hygiena SystemSURE (Set 2) Hygiena SystemSURE (Set 1) Biocontrol Lightening MVP (Set 1) Clean Trace NG Luminometer (Set 2) Clean Trace NG Luminometer (Set 1) Biocontrol Lightening MVP (Set 1) Charm Science (Set 1) Charm Science (Set 1) Neogen Accupoint (Set 1) Swab Device Hygiena Ultrasnap Hygiena Snapshot SBC 1575 Hygiena Snapshot SPXL 1333 Hygiena Supersnap Hygiena Ultrasnap Hygiena Snapshot SBC 1575 Hygiena Snapshot SPXL 1333 Clean Trace -2.75 Lightening Pocketswab Plus Hygiena Snapshot CH 1616 Neogen Accupoint -2.75 -2.50 -2.50 -2.50 Order of best extraction (mean of lowest dilutions detected) -3.75 -3.75 -3.50 -3.25 -3.00 -3.00 -2.75

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Table 17 Compendium Ranks of ATP Hygiene Monitoring Swabs for ATP Detection, ATP Recovery from swabs, Microbial Extraction and Food Extraction Levels
ATP Limit of Detection Hygiena Supersnap Hygiena Snapshot 1333 Hygiena Snapshot 1575 Hygiena Ultrasnap BioControl Lightning 3M Cleantrace Hygiena Snapshot 1616 Charm Pocketswab Neogen Accupoint ATP Recovery from Swab BioControl Lightning (100%) Hygiena Ultrasnap (93%) Charm Pocketswab (57%) 3M Cleantrace (52%) ATP Extraction from Microbial Cultures Hygiena Supersnap Hygiena Snapshot 1575 Hygiena Snapshot 1333 3M Cleantrace Hygiena Ultrasnap BioControl Lightning Hygiena Snapshot 1616N Charm Pocketswab Neogen Accupoint ATP Extraction from Foodstuffs Hygiena Ultrasnap Hygiena Snapshot 1575 Hygiena Snapshot 1333 Hygiena Supersnap 3M Cleantrace BioControl Lightning Hygiena Snapshot 1616N Charm Pocketswab Neogen Accupoint

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Monitoring Hand Cleanliness Using systemSURE II ATP Hygiene System


The importance of hand washing is a well-known fact. Reports indicate that inadequately washed hands are a factor in up to 40% of food-related outbreaks of illness. The low compliance rate (30-40% for Foodservice operators) is probably due to the lack of a rapid and simple method to assess whether effective hand washing has occurred, even if operators have been fully trained in correct hand-washing procedures. Hygiene monitoring based on ATP (adenosine tri-phosphate) bioluminescence is a simple method that can be used as part of a hand-washing-monitoring program to obtain results in real time. ATP is the universal energy molecule found in all living cells. The combination of ATP with the enzyme luciferase produces light that can be measured in a luminometer. The amount of light is proportional to the amount of ATP and is expressed in Relative Light Units (RLUs). The greater the level of ATP, the higher the RLU value, the dirtier the hand. Measurement of ATP can be used: a) during induction training to show the effectiveness of good hand-washing technique and b) to monitor efficacy of hand washing by swabbing clean hands immediately after washing (before hands come into contact with anything). Our validation data suggests that a reduction in ATP levels of greater than 75% is achievable following effective hand washing (requires two samples, one before and one after cleaning -- data not shown). For routine monitoring, Hygiena set a single Pass/Fail limit that would only require a single swabbing device per employee. Company employees washed their hands with soap and water for approximately 20 seconds and dried them with paper towels. The palm of the dominant hand* was swabbed using Ultrasnap and the devices measured in the systemSURE II luminometer. A Pass/Fail limit of 60 RLU was used. If the result was higher than 60 RLU, the volunteer was asked to rewash his/her hands for retesting.

Procedure:

Table 1. Routine monitoring of the ATP levels on employees hands immediately post washing.
Volunteer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 RLU Result 7 88 23 21 245 19 23 112 24 72 24 130 28 16 30 20 36 34 25 27 Action Yes/No No Yes No No Yes No No Yes No Yes No Yes No No No No No No No No Retest RLU 21 61a 14 15 81a -

* Only the dominant hand was swabbed for each volunteer a Bold = failed results and " "= retraining of hand-washing procedure required

For assessment of ATP levels immediately after hand washing, we found that there is a minimum RLU level that is attainable using an effective washing technique (see below). Studies show that the RLU value after hand washing is almost always below 100 RLU and below 60 RLU in most cases. Hygiena recommends setting a realistic Pass/Fail limit depending upon individual circumstances; e.g., type of food being handled, frequency of hand washing and type of soap/sanitizer used. Soaps vary in their effectiveness in reducing ATP levels. Before introducing an ATP hand-washing program we recommend testing the soap used before and after a thorough correct hand-washing procedure (minimum of 75% reduction in ATP level required). Hand-Washing Procedure It is a published fact that people tend to wash their hands in such a way that soiling and transient microorganisms are not removed from all areas of the hands equally. Hands should be washed frequently and thoroughly throughout the workday, especially after they have been exposed to sources of contamination. Proper hand-washing technique includes: Use warm water, sufficient amounts of soap/cleanser, and wash for 20-30 seconds Wash up to the forearms Use a nail brush to clean under fingernails Rinse with hands opened down into the sink Dry hands and arms thoroughly Use the paper towel to turn off the water and discard

Wash Hands After Blowing nose Coughing/sneezing Restroom and coffee breaks Personal grooming Touching unsanitary surface

For More Information Visit: www.hygienaUSA.com

Final

Hygiena International Ltd

Press Release HYG06

PRESS RELEASE HYG06

SYSTEMSURE HELPS IN FIGHT AGAINST HOSPITAL ASSOCIATED INFECTION


Hygiena International Ltd have joined forces with the North Tees and Hartlepool NHS Foundation Trust to provide them with a new weapon for their armoury in the fight against healthcare associated infections (HAI).

The Hygiena SystemSURE Plus ATP monitoring system is now being widely used by Trust Quality Monitoring Officers, Ward Matrons and Domestic Supervisors to check the cleanliness of patient areas within the hospitals. Previously, the Trust has relied wholly on visual inspections of cleaning standards; now the handheld, lightweight Hygiena instruments can provide a numerical result in seconds to show how clean or dirty a surface is. This enables the Trust to monitor the cleaning effectiveness of both the hospital environment and of the many types of patient equipment in use.

The Hygiena SystemSURE Plus instruments were originally introduced by the Trusts Infection Prevention and Control Department and have been used for handwashing training for several years. Use of the tool has now spread throughout both hospitals and staff and Managers are happy to use it to help eliminate the risk of infection in their areas.

It is a powerful tool, which has widespread applications throughout the healthcare sector to monitor areas such as Sterile and Catering Services Departments.

Press release Hygiene Monitoring Press Release - 30th July 2008 - 2 (Page 2 of 4)

A monthly programme of hygiene inspections using the Hygiena SystemSURE Plus commenced within North Tees and Hartlepool NHS Trust in May 2007 and every clinical inpatient area now has routine swabbing undertaken on a monthly basis.

The Trust has also been proactive in using the handheld instruments for other more diverse purposes such as swabbing clogs within Theatre, Doctors pens and stethoscopes and the pods that are used to deliver various goods between wards and departments within the Trust.

The palm sized SystemSURE instrument works by detecting the levels of adenosine triphosphate (ATP), a biochemical found in all living organisms and biological residues. If ATP is detected on a cleaned surface it means that the cleaning was not effective and the surface is potentially a hazard for the spread of germs. The ATP method is quick, simple and easy to do. A swab is taken of the area to be tested and is then inserted into the hand held instrument. The result is interpreted automatically and can be displayed within a matter of seconds as a simple pass, caution or fail display.

The Trusts Quality & Performance Manager Sue Shannon said, We are delighted to be working with Hygiena to introduce this system. The ATP monitor provides immediate feedback on the efficiency of cleaning and assists us in identifying areas that could have previously been overlooked; such as the underside of tables and down the side of chairs, where it is difficult to physically see contamination.

The results give us the evidence we need as a Quality Monitoring Department to tackle any problem areas and to produce reports for Ward Matrons on the cleanliness levels in their

Press release Hygiene Monitoring Press Release - 30th July 2008 - 2 (Page 3 of 4)

areas. More importantly it enables the Domestic Services Department to provide assurance that areas are as clean and as germ free as possible following cleaning, which is something we were previously unable to do, having relied purely on visual inspection.

We have also used the hygiene devices to check the efficiency of various methods of cleaning. As a result of this, we have invested heavily in microfibre technology over the past twelve months as it has proven to be the most efficient method of cleaning virtually every patient contact surface.

The Hygiena SystemSURE Plus is successfully marketed throughout 80 countries worldwide. Combined with their Ultrasnap sampling devices, it has become the premier system for hygiene monitoring not only in the medical and healthcare markets, but also in food, cosmetics and pharmaceutical manufacture, together with water treatment when combined with their Aquasnap sampling device. The instrument incorporates photodiode technology, and offers up to an 80% saving compared to competitors equipment. Simple keypad operation with LCD screen display is combined with storage capacity for 250 sites, a choice of 20 sampling plans and up to 50 named system users.

Further information is available from: Hygiena International Ltd, Unit 11, Wenta Business Centre, Colne Way, Watford, Hertfordshire WD24 7ND Telephone: 01923 818821 e-mail: enquiries@hygiena.net Fax: 01923 818825 www.hygiena.net

(approximately 550 words including photographic annotation) Continued ..

Press release Hygiene Monitoring Press Release - 30th July 2008 - 2 (Page 4 of 4)

GENERAL INFORMATION SUPPLIER: PRESS RELEASE: HYG06 READER RESPONSE INQUIRIES TO: General Immediate Mr Martin Easter, General Manager of

Hygiena International Ltd


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Hygiena International Ltd


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Photograph of a Quality Monitoring Officer using a handheld device on a patient bedside surface to be inserted here please.

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HYGIENA INTERNATIONAL LTD PRESS RELEASE HYG06

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Title: Effective Date:

Effects of sanitizers on Ultrasnap ATP Sample Testing Device June 2001

Summary The majority of common sanitizers (5 l at working strength) do not significantly affect the results obtained with the Ultrasnap ATP sample testing device Ultrasnap should not be used where an acid sanitizer has not been completely removed by rinsing, as it may affect the signal

Purpose To determine the effects of low levels of sanitizers on the results from the Ultrasnap ATP sample testing device.

Procedure Sample Preparation: ATP (disodium salt) was diluted in sterile pyrogen-free water (PFW) from a certified stock solution (2x10-2 Moles) to a concentration of 2x10-7 Moles. 10 l of this dilution was pipetted onto the Ultrasnap swab tip. Sanitizers were diluted in water to their recommended working strength and 5 l amounts were pipetted onto the Ultrasnap swab tip (separately from the water or ATP solution). Assay methods Ultrasnap device activity was measured as follows: 1. Remove swab from swab tube 2. Pipette 10l sterile pyrogen-free water or ATP solution directly onto the centre of the end of each swab tip 3. Pipette 5l sanitizer (diluted to working strength) or water (as a control) onto another area of the swab tip 4. Replace swab tube and break the blue snap valve in two directions 5. Squeeze reagent reservoir twice to dispense the reagent 6. Shake gently to bathe the swab in the reagent for 10 seconds 7. Measure activity by inserting the device in to the luminometer

This document and its contents are confidential and the exclusive property of Hygiena LLC. This document is not to be reproduced in any form whatsoever, without prior written permission from Hygiena LLC.

Materials
Ultrasnap devices: Validation Batch Luminometer: Prototype systemSURE II Sanitizers: Neutral surfactants, slight free caustic Quat and surfactant Quat and Glutaldehyde Alcohol, Quat and Biguanide Amphoteric Neutral surfactants, free caustic Free caustic Neutral surfactants, phosphoric acid Hydrochloric acid ATP standard Lot Number FA-05-15-01-1 Serial Number A6CW145K Kleencare Detergent Panel: AF123 or Topmaxx 123 DS607 or Triquart Super DS620 DS646 DS696 or Triquart AM NF421 or Topmaxx 421 NN4488 or MIP Betol SF520 or Topmaxx 520 SN570 2 x 10-7 M

Results ATP Response to Sanitizers Effects The results from the Ultrasnap device are relatively unaffected by the majority of sanitizers evaluated which were chosen to include sanitizers incorporating a wide variety of active ingredients. Under the conditions of this experiment, some sanitizers gave a slight decrease in signal and other sanitizers a slight increase. The hydrochloric acid sanitizer at the concentration used in this experiment decreased the signal the most of those tested. However, it is not known whether this is a realistic test; sanitizers should be removed from a surface by thorough rinsing, and the amounts used in this experiment may be unnecessarily high. Table C6.1. Effects of sanitizers (5 l at working strength) on response to ATP for the Ultrasnap hygiene monitoring device. Results are expressed as a percentage of the corresponding no sanitizer control. Sanitizer Code / Name AF123 or Topmaxx 123 DS607 or Triquart Super DS620 DS646 DS696 or Triquart AM NF421 or Topmaxx 421 NN4488 or MIP Betol SF520 or Topmaxx 520 SN570 Active Ingredient Neutral surfactants, slight free caustic Quat and surfactant Quat and Glutaldehyde Alcohol, Quat and Biguanide Amphoteric Neutral surfactants, free caustic Free caustic Neutral surfactants, phosphoric acid Hydrochloric acid RLU (% control) 103% 110% 113% 73% 77% 87% 114% 79% 65%

This document and its contents are confidential and the exclusive property of Hygiena LLC. This document is not to be reproduced in any form whatsoever, without prior written permission from Hygiena LLC.

Figs. C6.1-2. Effects of sanitizers (5 l at working strength) on response to ATP for the Ultrasnap hygiene monitoring device. Graphs show individual results from 6 replicate tests.
Fig. C6.1. Ultrasnap: effect of sanitizers on ATP response
1200 1000 800

RU L

600 400 200 0 PFW AF123 5% DS607 1% DS620 1% DS646 Neat

Fig. C6.2. Ultrasnap: effect of sanitizers on ATP response


1400 1200 1000

RU L

800 600 400 200 0 PFW DS696 1% NF421 3% NN4488 1% SF520 3% SN570 1%

Conclusions The majority of commonly used sanitizers (5 l at working strength) do not significantly affect the results obtained with the Ultrasnap hygiene monitoring device Ultrasnap should not be used where an acid sanitiser has not been completely removed by rinsing, as it may affect the signal

This document and its contents are confidential and the exclusive property of Hygiena LLC. This document is not to be reproduced in any form whatsoever, without prior written permission from Hygiena LLC.

ADVERTORIAL

Rapid tests of cleanliness deliver improvements and value

ne of the top priorities of the NHS operating framework is improving cleanliness and reducing healthcare-associated infections. New technology is providing a way to simply measure the quality of cleaning, the delivery of service levels and to ensure value for money. This technology, known as ATP bioluminescence, provides instant results and valuable management information that supports the concept of continuous measurable improvement. Cleaning can mean different things to different people, anything from general tidiness to absolute sterility, so effective, consistent training is essential. The NHS Cleaning Manual recommends effective cleaning systems and procedures, and their implementation fulfils the requirements of the Health and Social Care Act and Care Quality Commission registration. The aims of the NHS Cleaning Manual are to provide best practice guidance on cleaning techniques and advice on defining responsibilities, scheduling work, measuring outcomes, and reporting and driving improvements. Teamwork and documentation are essential to provide assurance and evidence of due diligence, as it is sometimes said that if it is not written down then it did not happen. The NHS has previously relied on visual assessments of surface cleanliness, but judging cleaning efficacy this way is subjective, and of questionable validity. However, the new, revised version of the NHS Cleaning Manual now recognizes that hygiene monitoring by ATP bioluminescence can provide an additional tool to monitor the delivery of cleaning services. The ATP bioluminescence technology is a wellestablished technology with more than 30 years of proven use in several industries. It has been given category 1 status under the Rapid Review Panel, and is also used in sterile services department for cleaning verification of washer disinfectors and endoscopes. The SystemSURE Plus ATP detection systems (Hygiena International) are being used in over 200 hospitals for several applications, including as a post-cleaning verification and monitoring tool from wards, in sterile service departments and catering facilities. It also makes a very good training and awareness tool for hand hygiene. It is simple and easy to use for nontechnical staff everywhere, and most importantly, it has provided an objective yardstick to yield quantitative numerical data about cleaning levels attained. In a case study conducted in the North Tees and Hartlepool NHS Foundation Trust shown on Health Exec TV (and also seen at http://www.hygiena.net/ind-healthcare.html), Kevin Oxley (Director of Operations) explains that they have introduced the Hygiena SystemSURE Plus to help combat the increase in HCAI:
We are seeing a growth in antibiotic-resistant bacteria and therefore we need to be able to validate our cleaning process to ensure that we can stop the spread of infections. Our cleaning scores have certainly improved since the introduction of the Hygiena SystemSURE Plus, and we have seen a corresponding decrease within the number

Housekeeping

Infection prevention and control

CQC Matrons

of infections in our patients, so we feel strongly that its helping combat the increase that we are seeing elsewhere within the NHS. The system is being used across the Trust by our quality monitoring officers, by our ward matrons and by our domestic supervisors, so that we can have instantaneous feedback on the standards of cleaning at ward level. The reports generated by Hygiena SystemSURE Plus are issued to all the relevant people, e.g. nursing manager and matrons, so that we can all work together as a team to rectify any problems that occur.

Working with link workers, Emma Davis (infection prevention and control nurse) said that the SystemSURE Plus:
highlighted to staff on the wards that it is actually really important they clean the equipment regularly and with the appropriate products. SystemSURE Plus is also incorporated into our hand hygiene training.

It is now possible to detect invisible contamination, and have rapid meaningful information to enable managers to monitor the delivery of cleaning services and ensure value for money. Effective cleaning is a keystone of infection control, yet it is frequently taken for granted and viewed as burdensome on finances. However, it has been shown that providing effective cleaning services reduces infection rates and produces a costbenefit analysis of >56 000 per ward, per annum (Rampling et al, 2001). The cost of failure is not only measured as human suffering and additional medical costs, but there are also severe financial penalties for Trusts that can run into millions. Getting it right makes good sense: clinically and economically.
Rampling A, Wiseman S, Davis L et al (2001) Evidence that hospital hygiene is important in the control of methicillin-resistant Staphylococcus aureus. J Hosp Infect 49(2): 10916

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