MICROBIOLOGICAL VALIDATION PROGRAMME FOR SRCL CLINICAL WASTE TREATMENT AUTOCLAVE PROCESSING METALS (SINGLE USE DEVICES) AT LEEDS

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1 MICROBIOLOGICAL VALIDATION PROGRAMME FOR PROCESSING METALS (SINGLE USE DEVICES) AT LEEDS M G Holliday MBA, PhD, MSc, CSci, FIBMS. MCIWM. F InstLM. Microbiology Department Freeman Hospital Newcastle upon Tyne NE7 7DN UK Dec 2014 Prepared for: SRCL Ltd SRCL/MVP/LEE/MGH/1214 Ver2 M G Holliday 1 of 24

2 EXECUTIVE SUMMARY The SRCL clinical waste autoclave unit at Leeds has proven to be capable of achieving and surpassing the required level of bacterial inactivation of metals waste when tested to EPR 5.07 protocols. The required inactivation level, (>4 log 10 inactivation of G stearothermophilus spores equating to STAATT level III inactivation) was met, and >6 log 10 inactivation of G stearothermophilus spores (equivalent to STAATT level IV) was demonstrated in 91.67% of individual tests. The SRCL clinical waste autoclave unit at Leeds can successfully and reproducibly heat disinfect clinical waste comprising single use metal instruments etc. to the required degree whilst operating under the stated operating parameters of 121 to 129 C with a holding time of 15 minutes and processing approximately 300 to 330 kg of waste per cycle. M G Holliday 2 of 24

3 1) INTRODUCTION The following testing programme was carried out for SRCL Ltd as a Microbiological validation of treatment programme to assess the effectiveness of the SRCL Clinical Waste treatment autoclave at Leeds in achieving the required level of Microbiological inactivation standards under normal operating conditions when processing metals waste. The metals waste is source segregated at the producer sites and is classified as infectious clinical waste arising from human healthcare and/or research (or potentially as or if the waste arises from veterinary or municipal sources). For validation of the efficacy of treatment, current UK Environment Agency (EA) 1,5 and NHS Estates (HTM 2075) 2 guidelines confirm that the levels of inactivation defined by the State and Territorial Association on Alternate Treatment Technologies (STAAT) 3 are accepted as valid, and clinical waste treatment facilities in the UK should meet at least level III inactivation criteria. Level III inactivation requires the demonstration of a 4 log 10 reduction in numbers of Bacillus atrophaeus (subtilis) or Geobacillus ( Bacillus) stearothermophilus spores 1,2,3,4,6 while level IV requires a 6 log 10 reduction in numbers of Geobacillus stearothermophilus spores 1,2,3,4. As the treatment system is an autoclave, G stearothermophilus was used in the validation tests. 2) SPECIFIC REQUIREMENTS FOR TESTING The EA guidance EPR 5.07 requires the following 5 : 1.Your validation report shall, as a minimum, contain the following information: a) a description of the treatment process and the parameters tested during validation b) the waste types and quantities included in each test c) who participated in the testing, their roles, and when the testing was done d) the site validation methodology and outline laboratory analytical methods e) parametric records from the treatment device f) test organism documentation/certification 2. Ensure that your report demonstrates that g) the STAATT level III criteria are achieved for the worst case scenario challenge load h) parametric controls, and procedures for real-time monitoring and assessment of treatment outputs, are in place and can be related to microbial efficacy M G Holliday 3 of 24

4 i) routine monitoring procedures, where they differ, are effective and have been proven comparable to validation procedures during site commissioning. In addition EPR 5.07 requires 5 : The spore species, strain and certification must be appropriate for the treatment process. For thermal processes the Level III tests must be performed using either Bacillus atrophaeus OR Geobacillus stearothermophilus (as appropriate). Level IV tests should be performed using Geobacillus stearothermophilus. Employ a single batch number of spore strips/spore suspension during commissioning. Where spore strips are used, each must contain 1 x 10 6 spores. For thermal processes the spores must have a certified thermal D-value 1.8 minutes at 121 C wet heat (Geobacillus stearothermophilus) at 160 C dry heat (Bacillus atrophaeus) Spores with Lower D-values must not be used. For thermal processes, the spores shall always be supported by the parallel use of thermal indicator strips (time and temperature) or multi-point thermal data loggers colocated in the waste load. The time/temperature combination used must be indicative of the required microbial inactivation being achieved All the above points will be addressed by this validation programme. Description of the treatment process and operating parameters. The Leeds autoclave is a batch autoclave (Figure 1) which accepts metals waste (single use instruments etc) which are transferred by an automated system from reusable biosystems sharpsbins into two autoclave carts. The carts are placed inside the autoclave and the treatment process operates at above 121 C with a holding time of 15 minutes achieving temperatures of 128 to 130 for much of the time. The resulting metal can then be sent for recycling. Waste Types and quantities Metals wastes (single use instruments such as scissors, stitch cutters, handles of instruments such as laryngoscopes) are processed by the system (Figure 2). Typical loads are 150 to 170 kg per cart or a total of 300 to 350 kg per load. The number of spore strips to be tested per cycle and per cycle format are also defined in EPR 5.07 as follows: M G Holliday 4 of 24

5 Single Load Capacity (Kg) Continuous throughput (Kg per Hour) recovered per cycle or collection. recovered for each cycle format 0-10 kg kg kg kg kg >750 kg retained as controls The Leeds autoclave has a throughput of approximately 350 kg, therefore a minimum of 8 spore strips per cycle and 24 strips per unit is required. As this system does not pre-macerate the waste, EPR 5.07 requires the use of worst case challenge loads which are designed to place the spore tests inside the toughest items commonly found in the waste loads. In nonselected Clinical Waste this is generally taken to mean that spore strips should be fixed in the centre of filled, sealed suction canisters and chest drains, however as these items can not be placed inside sharpsbins, they can not form part of these loads and are therefore inappropriate. EPR 5.07 suggests the use of other challenging items where heat penetration may be inhibited (for example lengths of tubing, inside syringe bodies in sealed sharps boxes etc) can be used to reflect the types of items found in the loads. For this study, spore strips were wrapped in cotton wool and placed inside sterile laryngoscope handles which had the batteries removed (Fig 3). The caps were then screwed back onto the handles. These were felt to be the worst case challenge load that could be encountered in a normal waste load. 3) AIMS AND OBJECTIVES To investigate whether the SRCL Clinical waste treatment autoclave at Leeds can meet the required STAATT level III inactivation standard as specified in EPR when processing metals waste. M G Holliday 5 of 24

6 4) GENERAL During Microbiological validation and testing, it is suggested that a suitably qualified and experienced independent Microbiologist is present to supervise the process, ensure the validity of the testing and to advise on any factors that might adversely affect the outcome of the tests 5. It is also necessary that any laboratory should have suitable competence or accreditation. Dr M Holliday was the microbiologist on-site during the testing period and supervised all stages including the preparation, loading, retrieval, storage and transport of all test pieces and the laboratory analysis and result interpretation. The testing was carried out in the Microbiology Department Freeman Hospital, which has full Clinical Pathology (UK) Accreditation, and has carried out in excess of 50 waste treatment validations. 5) OPERATING PARAMETERS DURING THE The testing took place on 15 th December During the testing programme, the operating parameters of the system were 129 C (2.65 BAR) with a holding time of 15 minutes, with 3 waste loads of 308.2, and kg of metals waste respectively representing the anticipated normal content. The operating parameters were checked visually during each cycle to ensure they were as anticipated. An example of the parameters recorded during one of the runs are shown in Table 6. 7) TEST PIECES a) Spore strips Commercially available G stearothermophilus spore strips containing a certified population of 2.6 x 10 6 spores per strip and with a D 121 -value of 2.2 were used to demonstrate the required log reduction in numbers. These were all from a single batch number as outlined in EPR S5.07 (Appendix 2). b) Thermal Integrator Strips Browne TST Thermal Integrators (Steris Ltd) with published setpoints of 12 minutes at 121 C were used. When these conditions are met, the colour changes from Yellow to Blue. These parameters are be indicative of the required microbial inactivation being achieved as the spores used are stated to survive 9.7 minutes at 121 C but be completely killed by 22.9 minutes at 121 C (Appendix 2). M G Holliday 6 of 24

7 8) TEST METHODOLOGY a) Validation testing In each test load, one spore strip and one thermal integrator were placed in each of 8 net bags. These were buried in the centre of the waste load (4 per cart). The net bags were attached to tennis balls by a length of wire. The tennis balls were left outside the autoclave cart to aid identification and recovery of the spore strips after treatment. One spore strip and one thermal integrator wrapped in cotton wool were also placed inside each of 8 empty laryngoscope handles with the bottom screwed back on as worst case challenge loads (4 per cart) (Figure 3). These were attached to tennis balls and buried in the waste in the same way as above (Figure 4). This resulted in 8 spore strips and thermal integrators in net bags and 8 spore strips and thermal integrators in handles per autoclave cycle. When the autoclave was fully loaded with the 2 carts of metals, the door was closed and the process was started. The process is automatic and is carried out under PLC control. When the process cycle was complete, the door was opened and the carts were removed, the spore tests and thermal integrators were recovered by tracing the wires down through the waste to the net bags and handles. A new cycle of spore tests was only commenced once all the previous spore tests had been retrieved from the system. The test spore strips and thermal integrators were given unique identification numbers and were placed inside sealed plastic bags for transport back to the laboratory and analysis as described in Appendix 1. The above tests were undertaken on 3 separate cycles of the autoclave unit. Controls Two unexposed G stearothermophilus spore strips were held beside the autoclave to act as untreated controls for each test cycle 1 and uninoculated TSB broths were used as negative field controls This resulted in a total of 16 spore tests plus 2 controls per cycle giving a total of 48 spore tests and 6 controls overall. The required level of inactivation will be a 4 log 10 reduction in numbers of G stearothermophilus spores equating to STAATT Level III inactivation. M G Holliday 7 of 24

8 100% of the test strips recovered must be analysed and the mean number of spores recovered from the untreated control strips must be compared to the mean number of spores recovered from the test strips. The following criteria represent the minimum standard that must be achieved: 1) The mean recovery from the control strips must be equal to or greater than log4 2) The difference between the mean recovery from the control strips and the mean recovery from the test strips must be equal or greater than log4. Data Analysis of the spore test results follows the procedure outlined in EPR 5.07, Annex 1 (Appendix 3). M G Holliday 8 of 24

9 9) RESULTS Controls All 6 untreated control strips grew G stearothermophilus at a population of 10 6 per strip. The results are shown in Table 1. Table 1 Results of quantitative counts on control spore strips Cycle Count in cfu Log a a a This resulted in an average recovery of 6.00 x log 10 spores Of the 48 spore strips submitted for testing, all 48 were recovered. 48 Thermal integrators were also recovered Results are shown in Tables 2, 3, 4 and 5. Table 2 Results of Spore Tests 1 st cycle Spore strip No Spore Carrier Cfu/mL of G stearothermophilus recovered Log count of G stearothermophilus recovered Log 10 kill of G stearothermophilus demonstrated 1 Net bag 0 0 >6 log 10 2 Net bag 0 0 >6 log 10 3 Net bag 0 0 >6 log 10 4 Net bag 0 0 >6 log 10 5 Net bag 0 0 >6 log 10 6 Net bag 0 0 >6 log 10 7 Net bag 0 0 >6 log 10 8 Net bag 0 0 >6 log 10 9 Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log 10 M G Holliday 9 of 24

10 Table 3 Results of Spore Tests 2 nd cycle Spore strip No Spore Carrier Cfu/mL of G stearothermophilus recovered Log count of G stearothermophilus recovered Log 10 kill of G stearothermophilus demonstrated 1 Net bag 0 0 >6 log 10 2 Net bag 0 0 >6 log 10 3 Net bag 0 0 >6 log 10 4 Net bag 0 0 >6 log 10 5 Net bag 0 0 >6 log 10 6 Net bag 0 0 >6 log 10 7 Net bag 0 0 >6 log 10 8 Net bag 0 0 >6 log 10 9 Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle log Metal handle log 10 Table 4 Results of Spore Tests 3 rd cycle Spore strip No Spore Carrier Cfu/mL of G stearothermophilus recovered Log count of G stearothermophilus recovered Log 10 kill of G stearothermophilus demonstrated 1 Net bag 0 0 >6 log 10 2 Net bag 0 0 >6 log 10 3 Net bag 0 0 >6 log 10 4 Net bag 0 0 >6 log 10 5 Net bag 0 0 >6 log 10 6 Net bag 0 0 >6 log 10 7 Net bag 0 0 >6 log 10 8 Net bag 0 0 >6 log 10 9 Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log Metal handle 0 0 >6 log 10 Note: The Log 10 kill of G stearothermophilus demonstrated is determined by taking the log 10 of the test strip and subtracting it from the log 10 mean of the control strips. M G Holliday 10 of 24

11 Table 5 Summary of Spore test results Cycle number Spore carrier Spore strips and thermal integrators Recovered Thermal Integrators Pass at 121 C for 12 minutes Number of Spore strips >6 log 10 kill Number of Spore strips >4 log 10 kill Interpretation (meeting the requirement for log 4 inactivation) 1 Net bags PASS 1 handles PASS 2 Net bags PASS 2 handles PASS 3 Net bags PASS 3 handles PASS TOTAL strips showed no growth from the broth cultures indicating that all spores present had been inactivated, thus demonstrating > log 6 kill while the remaining 4 strips showed log 4 kill or greater. 44 of the 48 thermal integrators changed colour indicating that the setpoint had been achieved. Four thermal integrators failed to change colour. The spore strips that did not achieve log 6 kill were in the same four worst case challenge handles as the thermal integrators that failed to change colour. Table 6 Operating Parameters observed during Autoclave cycle 3 Time Temperature recorded : : : : : : : : : The cycle started at 12:05 was complete at 12:26 when the system commenced cooldown. This shows that the chamber was above 121 C for over 15 minutes and was above 128 C for more than 10 minutes. The visible parametric display is shown in Figure 5. M G Holliday 11 of 24

12 Data Analysis and interpretation 5 The following calculation follows the data analysis methodology specified in EPR 5.07 (Appendix 2). STEP EA S5.06 Annex 1 Section 1.6 CONTROL DATA 1 Control strips cfu , , , , , mean control recovery (XC) log10 (Xc) PASS CRITERIA log10 ( Xc) -4 = 2.0 (Level III criteria) 5 TEST DATA three test runs each with 16 strips 6 mean test colony recovery (XT) Std Deviation of results (σ) Upper 95% CI (Lu) (6.46+(1.96* 24.45) = Log10 of LU 1.74 It has been determined in step 4 that the Pass Criterion = 2.0. Log 10 Lu MUST be less than this figure. It has been determined in step 9 that the log 10 of the upper 95% confidence interval (log 10 Lu) of the spores recovered from the test runs = 1.74 The results from the test runs show that the log of the upper 95% confidence interval (Log 10 Lu) for recovered spores (1.74) is less than the pass criteria (2.0) Log10 (Xc) is greater than 5 so sufficient spores have been recovered for the results to be valid All but 4 thermal indicator strips have indicated that the required time/temperature parameters were achieved. STAATT Level III criteria have therefore been successfully demonstrated Where these criteria are passed then it is >97.5% probable that the worst case items present in any clinical waste will be treated to the minimum standard. M G Holliday 12 of 24

13 10) DISCUSSION The results in table 1 show that all of the extracted control G stearothermophilus strips had the organism extracted by the procedure to an average level of 6.00 log 10. This count can be used in quantitative tests for comparison with treated strips to determine the exact level of log 10 kill achieved if less than complete inactivation is achieved. In these tests, 44 of 48 (91.67%) test strips from the unit showed no growth demonstrating complete inactivation. As the strips had certified populations of 2.6 x10 6 spores per strip, the log 10 kill can be deemed equal to or greater than 2.6 x10 6 however for the purposes of the statistical data analysis in this report, a log kill of 1 x 10 6 (the log of the control strips minus the log of any growth of the test spores) was used. Three of the remaining spore strips grew a single colony each from the 1:100 dilution plates and demonstrated growth in the broth cultures. This gives a total count of 100 cfu and equates to a 4.0 log 10 kill. The last spore strip grew a single colony from the 1:10 dilution plate. This gives a total count of 10 cfu and equates to a 5.0 log 10 kill. All recovered strips were included in results and the data analysis shows that the pass requirements of EPR 5.07 have been fully met. This demonstrates that STAATT level III inactivation has been successfully achieved. 100 % of the individual spore tests demonstrated greater than 4 log 10 kill (representing STAAT level III standard) and % of the tests demonstrated >6 log 10 kill of G stearothermophilus which equates to STAATT level IV kill and provides a large margin of safety over the level required. It is worth noting that all the spore strips that showed less than log 6 kill were in the worst case challenge containers along with the thermal integrators that did not change colour. The fact that log 4 spore kill was demonstrated in these containers shows that the thermal integrators do not correspond exactly with the spore results. Thermal indicators appear to be a good indicator of less than optimal spore killing, even when this is equal to or greater than log 4 as they fail to change colour before the spore strips show failure and are therefore a more sensitive early warning of potential performance problems. Some of the laryngoscope handles used as worst case challenge containers had the caps screwed down extremely tightly after treatment and required the use of a wrench to remove them. It is suggested that these may be providing an unacceptably difficult challenge to the system, although they appeared to be a good choice in planning the testing programme. M G Holliday 13 of 24

14 Laryngoscope handles are used to hold the batteries for the unit and are tightly sealed in use to prevent access of liquids. As the units are never opened in areas where they could become contaminated by infectious substances it is argued that they should not be used as worst case challenge containers in future. As can be seen from Figure 2, most of the handles normally occurring in the waste were missing their screw on caps so it is suggested that future validation tests should use spore strips wrapped in cotton wool inside handles without the caps as a more realistic worst case challenge. Routine Monitoring For routine monitoring of the process, it is suggested that spore strips in net bags be used as test pieces in the manner utilised in this report. 12) CONCLUSION The SRCL clinical waste autoclave unit at Leeds has proven to be capable of achieving and surpassing the required level of bacterial inactivation of metals waste when tested to EPR 5.07 protocols. The required inactivation level, (>4 log 10 inactivation of G stearothermophilus spores equating to STAATT level III inactivation) was met, and >6 log 10 inactivation of G stearothermophilus spores (equivalent to STAATT level IV) was demonstrated in 91.67% of individual tests. The SRCL clinical waste autoclave unit at Leeds can successfully and reproducibly heat disinfect clinical waste comprising single use metal instruments etc to the required degree whilst operating under the stated operating parameters of 121 to 129 C with a holding time of 15 minutes and processing approximately 300 to 330 kg of waste per cycle. M G Holliday 14 of 24

15 APPENDIX 1 LABORATORY PROCESSING OF SPORE STRIPS On receipt at the laboratory, all control and test spore strips envelopes were aseptically peeled open and the strips transferred to 10 ml of sterile Tryptone Soya Broth containing Phenol Red (TSB). For validation processes, quantitative counts were carried out as follows to determine the exact degree of heat inactivation achieved where total inactivation (no growth) was not achieved. Spore strips in TSB were vortexed 3 times for 1 minute with a 10 minute soak between to elute spores from the strips. Serial dilutions of these were made in sterile distilled water. 1 ml of the original TSB broth and 100 ul of each dilution were added in duplicate to Tryptone Soya Agar (TSA) plates and spread across the surface (Quantitative count plates). These plates were used to count the number of G stearothermophilus spores that survived the treatment process and germinated. Quantitative count plates were allowed to dry and were then incubated at 55 C for 48 hours before being examined for the presence or absence of G stearothermophilus organisms. The number of colonies were counted and recorded The Tryptone Soya Broths were incubated at 55 C for 48 hours before being subcultured onto Tryptone Soy Agar plates. These plates were then incubated at 55 C for 48 hours before being examined for the presence or absence of G stearothermophilus organisms. The presence of any growth of G stearothermophilus from a test Spore strip equating to less than 4 log 10 inactivation will result in that test being designated a FAIL. Repeated or reproducible Fail results from the exposed strips are evidence of ineffective heat disinfection. The quantitative log 10 kill of spores from these strips can help identify the degree of ineffectiveness and can inform decisions to remedy this. The absence of G stearothermophilus growth is taken as complete inactivation of the original spore population (in this case 2.6 x 10 6 or log6) or equal to or greater than 4 log 10 inactivation (STAATT level III) from a test Spore strip will be designated a PASS result. If the required level of inactivation has been proven to be achieved, then the unit should be considered suitable for the treatment of clinical waste to make it safe. M G Holliday 15 of 24

16 APPENDIX 2 Spore Strip certification M G Holliday 16 of 24

17 Tryptone Soy Broth Certification M G Holliday 17 of 24

18 APPENDIX 3 DATA ANALYSIS 5 Appropriate measures for Microbial Disinfection Efficacy Spore Strips Control Data For the control data the following are required: 1. The number of spores recovered from each of the individual control spore strips is calculated in cfu 2. The mean number (XC) of spores recovered from the control strips is calculated in cfu, 3. The log10 of (XC) should be determined. 4. Subtract 4 from the log10 of (XC) to generate the pass criteria. The subtraction of 4 is used to calculate the 4 log10 reduction required to achieve the STAATT Level III criteria. (Where required, the pass criteria may be adjusted to (log10 (XC)-6)) Test Data For the combined test runs the following require determination: 5. The number of spores recovered from each individual test strip. 6. The mean (XT) number of spores recovered, 7. The standard deviation () of spores recovered, 8. The Upper 95% (Lu) confidence interval of (XT) (this will be approximated by XT+ 1.96), 9. The log10 of the Upper 95% (Lu) confidence interval of XT. (log10lu) (note if Lu = 0, then use 0 for log10lu ) This must include all the recovered test strips. If contamination is suspected you should either retest the sample or, if that is not possible, include the potentially contaminated sample results in the data analysis. Interpretation The following criteria represent the minimum standard that must be achieved: 10. The log10lu for each run must be less than or equal to the pass criteria 11. log10(xc) must be For thermal processes all thermal indicator strips should indicate that the required temperature time parameters have been achieved. Where these criteria are passed then it is >97.5% probable that the worst case items present in any clinical waste will be treated to the minimum standard. M G Holliday 18 of 24

19 REFERENCES 1) Environment Agency. Technical guidelines on Clinical Waste Management Facilities. Version 2.5. March ) Clinical Waste Disposal/treatment technologies (alternatives to incineration) Health Technical Memorandum NHS Estates ) Technical Assistance Manual: State Regulatory Oversight of Medical Waste Treatment Technologies. A Report of the State and Territorial Association on Alternate Treatment Technologies. April ) Slavik,NS Use of Germicides in Medical Waste Treatment. In: Chemical Germicides in Health Care. Ed. Rutala WA. Association of Professionals in Infection Control and Epidemiology, Inc. ISBN p ) Environment Agency. Clinical Waste EPR S5.07 Ver 1.1 Jan 2011.Annexe 1. Section 1.3 6) Henry S. Luftman and Michael A. Regits Applied Biosafety Vol. 13, No. 3, 2008 M G Holliday 19 of 24

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21 Appendix 2 Tryptone Soya Broth Certification M G Holliday 21 of 24

22 Figure 1 The Leeds Clinical Waste Autoclave Figure 2 Items encountered in a normal load of metals waste M G Holliday 22 of 24

23 Figure 3 The Laryngoscope handle used as worst case challenge attached to a tennis ball Figure 4 The Carts ready for loading into the autoclave with the test pieces buried in the waste and identified by the wire attached to tennis balls M G Holliday 23 of 24

24 Figure 5 Readout on the PLC screen of the autoclave showing pressure and temperature inside the chamber M G Holliday 24 of 24