Annual Report. 1 st April st March Laboratory Service: Toxoplasma gondii. Provider: NHS Highland

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1 Annual Report 1 st April st March 21 Laboratory Service: Toxoplasma gondii Provider: NHS Highland Author: Dr D. O. Ho-Yen Director Scottish Toxoplasma Reference Laboratory Date: May 21 1

2 1. Summary: Key Points 1. Clinical Pathology Accreditation (United Kingdom) A full CPA inspection took place on the 1 st and 2 nd July 29. The laboratory retained full CPA Accreditation. There were no Reference Laboratory noncompliances or observations. 2. Quality Assurance The high success rate in internal and external schemes demonstrates the quality of the service. 3.Activity The overall test activity was 9.7 % lower than predicted for the year. 4. Turnaround times 99.9 % of test results were available within the required turnaround time of 7 working days. 5. Positive Results A breakdown of the serology and clinical results has been included to reflect the work of the laboratory. 6. Finance The financial outturn had a 5.2% overspend due to AFC reviews which were not funded. 7. Agenda for Change The outstanding AFC reviews have finally been completed for Reference Laboratory staff. 8. Reference Laboratories Review The Reference Laboratory Review has been completed and our contract will be renewed for another 3 years. Details of the new contract have still to be agreed. 2

3 2 Activity data 2.1 Contracted activity Test Contracted number Actual number ELISA IgG ELISA - BAM DYE TEST ISAGA IgM IgA IgE IgG AVIDITY WESTERN BLOTTING TOXOREAGENT LATEX *AXSYM IgM PCR ISOLATION TOTAL *performed to support the IgG avidity test The overall test activity of 5741 was 9.7% lower than predicted for the year. This is lower than in previous years: 5977 tests in 21-2, 7226 tests in 22-3, 6933 tests in 23-4, 755 tests in 24-5, 69 tests in 25-6, 6628 tests in 26-7, 6317 tests in 27-8 and 6495 in This reflects the fall in the number of samples from 1946 to 1816 and the continued fall in ISAGA testing from a contracted level of 668 to 94 this year. The ISAGA is still essential for the Reference Laboratory but its use is now more correctly targeted to the investigation of symptomatic immunocompromised patients and congenital babies. The number of Western blots has increased from 2 last year to 12 this year. This allows direct comparison of mother and baby antibody patterns and is the preferred test for investigation of congenital toxoplasmosis 3

4 2.2 Activity by Health Board of referral Health Board No. of Samples (%) Current Year No. of Samples (%) Previous Year ARGYLL & CLYDE AYRSHIRE & ARRAN BORDERS DUMFRIES & GALLOWAY FIFE GRAMPIAN GREATER GLASGOW LANARKSHIRE LOTHIAN TAYSIDE HIGHLAND & WESTERN ISLES NORTHERN IRELAND SWANSEA REFERENCE UNIT (.) (.) 14 (.8) 18 (.9) 1 (.1) 6 (.3) 64(3.5) 71 (3.6) 16(.9) 7 (.4) 43(2.4) 29 (1.5) 63(33.2) 638 (32.8) 114(6.3) 98 (5. ) 252 (13.9) 278 (14.3) 97(5.3) 9 (4.6) 273(15.) 231 (11.9) 339 (18.7) 469* (24.1) (.) (.) 1816(1) 1946 (1) 27.7% fall in number of samples from Northern Ireland *represents test activity of 7.1% for current and 9.2% for previous year as they screen. Activity in previous years: 1318 samples in 21-2, 1645 samples in 22-3, 1842 samples in 23-4, 24 samples in 24-5, 192 samples in 25-6, 197 samples in 26-7 and 1948 in The pattern of referral from different Health Boards has been consistent. Northern Ireland activity has been maintained below the agreed level of 15%: 9% in 21-2, 9.5% in 22-3, 8.6% in 23-4, 9.4% in 24-5, 1.5% in 25-6, 8.8% in 26-7, 12% in 27-8 and 9.2% in

5 Positive Results Serology results Toxoplasma antibody Patient group No negative Equivocal Positive BMT (67.3%) 14 (9.3%) 35 (23.3%) Donor/Recipient (4.2%) 16 (15.7%) 45 (44.1%) HIV/ high risk/ (7.2%) 21 (6.7%) 67 (21.8%) immunocompromised Ocular 19 4 (36.7%) 13 (11.9%) 56 (51.4%) Pregnancy (47.8%) 118 (52.2%) Congenital (71.2%) 19 (28.8%) Other clinical (53.2%) 6 (1.3 %) 213 (43. 2%) No details (37.2 %) 5 (1.4%) 218 (61.4%) Total (52.6 %) 75 (4. 2%) 771 (43.5%) Clinical results a) Bone Marrow Transplants: 11 negative results make toxoplasma reactivation unlikely, 35 positive results makes reactivation a major consideration and 14 equivocal results need retesting. b) Donor/ recipient: in 86 cases mismatches can be avoided and in 16 equivocal cases there needs to be further testing. c) HIV/ High Risk/ Immunocompromised: in 219 cases reactivation is not a problem, in 67 cases reactivation has to be seriously considered and in 21 equivocal cases further testing is required. d) Ocular: in 4 cases the negative serology is incompatible with congenital ocular toxoplasmosis, in 56 cases results supports a diagnosis of congenital toxoplasmosis and in 13 cases there needs to be repeat serology. e) Pregnancy: 18 cases there is no evidence of past infection and mothers must be given advice to avoid infection and in 118 cases there is evidence of past infection and the mother is immune or further tests are required to exclude current infection. f) Congenital: 47 cases there is no evidence of congenital infection and 19 cases there is supporting evidence. g) Current infection: 26 cases; 22 in Scotland and 4 in Northern Ireland 5

6 3 Quality Assurance Report 3.1. Turn around times by test category Test 1. ELISA IgG ELISA BAM DYE Test No No (%) with turnaround time in days 2 days < 7 days > 7 days 145 (69.4) 46 (3.6) 151 (69.8) 454 (3.2) 1871 (88.9) 234 (11.1) TOTAL (77.5) 1149(22.5) 2. IgG Avidity ISAGA IgM IgA IgE Mast Latex Axsym-IgM (17.7) 5 (11.1) 5 (9.5) 23 (3.1) 46 (18.9) 15 (82.3) 38 (88.9) 46 (9.5) 54 (69.9) 198 (81.1) TOTAL (18.8) 441 (81.2) 3. Western blotting 12 4 (33.3) 8 (66.7) PCR 71 29(4.8) 41 (57.7) 1 (1.4) * Isolation TOTAL (71.4) 1639 (28.6) 1 (.1) 1. First line serological tests 2. Secondary confirmatory tests 3. Tests being monitored * Not applicable minimum time 4-6 weeks For first line tests 77.5% of results were available within 2 days. For the dye test 88.9 % of results were available within 2 days. Overall, 99.9% of results were available within the contract level of 7 working days. 6

7 3.2. Internal audit (Appendix A) Horizontal audit: 2 audits performed: sick leave and workload statistics Vertical audit: 1 audit was performed Examination audit: 4 audits performed: cell culture reagent preparation, preparation of PCR samples, Mast Toxoreagent, use of Class 2 Safety cabinet Internal Quality Assurance 1 samples: recycled in1 distributions of 1 sample. 32tests: ELISA IgG (1), BAM ELISA (1), Dye test (1), AxSym IgM (2) There were no differences in test selection, results or interpretation Clinical Effectiveness These were joint audits with the Virology department An audit to evaluate a commercial Alert RT PCR kit. The system is sensitive and easy to use but would be very much more expensive than our in- house assay. The system does offer an internal extraction control which would be a useful addition to our assay An audit to review inhibition in PCR tests for Toxoplasma gondii and Borrelia burgdorferi. Inhibition was a well recognized problem with nested PCR and consequently samples were tested neat and at 1:1 dilution. This audit confirmed that inhibition was not a problem with real time PCR and testing at 1:1 was not required. Samples are now tested neat, in duplicate, without additional cost and test performance is easier. This has improved PCR testing An audit to replace diluters with automatic pipettes for dilutions in the toxoplasma dye test. Diluters have been used for > 2 years but are no longer manufactured and cannot be replaced. It is essential that we move to a sustainable alternative. The test is being recalibrated An audit to compare preparation of biotin labelled toxoplasma with and without use of a shaking water bath. A shaking waterbath has always been used to prepare this antigen but the waterbath is failing and will be very expensive to replace. Alternatives are being investigated. 7

8 3.3 Accreditation Schemes ( Appendix B) Name of Scheme Current status Date/type of last visit Clinical Pathology Full Accreditation 1 st / 2 nd July 29 Accreditation (UK) CPA Reference: Full Inspection Ltd 492 Date /type of next visit Surveillance visit Date to be arranged There were no Reference Laboratory non compliances or observations. The letter and certificate of CPA Accreditation was received in November External Audit Schemes (Appendix C) UK NEQAS Toxoplasma IgG Serology 18 samples, 3 distributions of 6 samples Pre and post distribution testing 18 tests: ELISA IgG (36), Dye test (36), Mast Toxoreagent (36) Success rate: 1% UK NEQAS Toxoplasma IgM Serology 8 samples, 2 distributions of 4 samples. Pre and post distribution testing 32 tests: BAM ELISA IgM (16), AxSym IgM (16) (* ISAGA not done because of kit supply problems) Success rate: 1% National Toxoplasma Reference Unit Serology 2 samples, 2 distributions of 1 samples 116 tests: ELISA IgG (2), Dye test (2), Mast Toxoreagent (2), BAM ELISA IgM (2), AxSym IgM (15), ISAGA IgM (6), ISAGA IgA (6) IgG avidity (9). There was 1 discrepancy: 1 ISAGA IgM. Success rate: 99.2% National Toxoplasma Reference Unit PCR 8 samples, 2 distributions of 4 samples (1, 1, 1, toxoplasma tachyzoites) There was 1 failure to detect a sample containing 1 tachyzoites Success rate: 87.5% Health and Safety Executive Inspection Containment Level 3 facilities were inspected on 19 th November 29. There were no non- conformities. 8

9 3.5 Incidents and Complaints A sample received on Friday afternoon was not entered into the computer system until Monday morning. Booking in procedures have been reviewed to ensure that all samples are logged on the day received. 3.6 Equality and diversity impact assessments Paul Nairn Service Planning Manager and Jean Chatterton Principal Clinical Scientist had an Equality and Diversity Impact Assessment meeting on 12th March 21. The EQIA checklist was completed satisfactorily and identified no requirement for change. A copy was sent to Dr Anne Eastaway to report back to the EQIA group. 4. New developments 4.1 We have demonstrated that inhibition was not a problem with real time PCR and routine testing of samples neat and at a 1:1 dilution was not required. Samples are now tested neat, in duplicate, without additional cost and test performance is easier. Seeded samples are still tested to identify inhibition. Any samples which inhibit can subsequently be tested at a 1:1 dilution. This has simplified and improved PCR testing HPS Reporting From 1 st April 21 positive cases will be reported by ECOSSE. 5. Future developments 5.1 Preparation of antigen for the in-house IgM ELISA from cell culture tachyzoites. Small amounts of antigen have been prepared but the quantity and concentration must be increased to support the routine testing. A method to produce the required numbers of tachyzoites is being developed. 5.2 The development of a Toxoplasma NEQAS PCR scheme with the Quality Assurance Laboratory at Colindale and the National Institute of Biological Standards (NIBS) is continuing. It was hoped that a standard had been prepared and samples would soon be distributed for testing. 5.3 Assessment of immunoblotting to improve the serological diagnosis of ocular toxoplasmosis. 9

10 6 Teaching 6.1 Internal Staff Training Type of training Details Numbers attending Practical (i)training in Reference Laboratory Tests for the new Clinical Scientist (ii) Refresher training for BMS staff rotating 1 2 between Virology and Toxoplasma Discussion/ Monthly Clinical Effectiveness Meeting 12 Audit Problems/ planning Monthly staff meetings 6 Presentation/discussion Journal Club 8 th April Antibody response of HIV infected patients to latent, cerebral and recently acquired toxoplasmosis 6.2 Individual Placements Visitor Grade Speciality Base Place of Work Duration of placements Liz Dickson Trainee Clinical Scientist Microbiology 6.3 External Training Microbiology Raigmore Hospital Inverness 17 th -21 st August 29. Type of training Topic Numbers attending NEQAS Teaching Sheet May 29 Toxoplasma in bone marrow transplant patients All participants NEQAS Teaching Sheet October 29 NEQAS Teaching Sheet January 21 Risk factors for Toxoplasma gondii infection NEQAS Teaching Sheet All participants All participants 1

11 6.4 Publications (i) Chatterton JMW, Evans R, Ho-Yen DO. Ocular toxoplasmosis- a Laboratory View. HPS Weekly Report Current note HPS Weekly Report 31 st March 21. Full article diagnosis of ocular toxoplasmosis.pdf. (ii) Chatterton JMW, McDonagh S, Ho-Yen DO. Toxoplasma tachyzoites from cell culture are more appropriate in some situations. J Clin Path. Published on line 1 st April 21. (iii) Manser MM, Chatterton JMW, Guy E, Francis J, Holliman R, Ho- Yen DO, Johnson J, Chiodini PL. The United Kingdom National External Quality Assessment Scheme for Parasitology: Toxoplasma Serology Subschemes. J Clin Path (submitted for publication) (iv) UK NEQAS Toxoplasma Scheme Teaching Sheets May 29 Toxoplasma in bone marrow transplant patients October 29 Risk factors for Toxoplasma gondii infection January 21 Toxoplasma IgG avidity testing 7 Research 7.1 Investigation of effect of long term maintenance of Toxoplasma gondii tachyzoites in cell culture. 7.2 Use of cell culture- derived tachyzoites in BAM-IgM ELISA and ISAGA tests. 7.3 Development of cell culture techniques for isolation of toxoplasma. 7.4 Investigation of adaptation of Toxoplasma gondii tachyzoites to cell culture. 8 Staffing 8.1 The outstanding Agenda for Change reviews for the BMS2 and Clinical Scientist posts have finally been completed and the jobs have been matched against a higher band. This has been a long and difficult process and the support from HPS has been much appreciated by staff. 1 Summary and conclusions The Scottish Toxoplasma Reference Laboratory test activity was lower than predicted for the year (5741 tests, planned 6361). This is largely due to the fall in ISAGA testing from the 668 planned to 94. Successful retention of full CPA Accreditation and success in internal and external quality assurance schemes confirms the quality of the service. 11

12 11. Appendices Appendix A Internal Audit Exception Reports/ Risk Register 1 Error log 1.1 Two reports were sent to the wrong location. Staff were reminded to check report addresses. 1.2 One report was issued with an incorrect comment. The laboratory was immediately informed and an amended report issued. 2 Risk register 2.1 An updated risk log is attached. There is one addition: the Medipath information management system. 2.2 Incidents Failure to recover toxoplasma cultures from liquid nitrogen. The laboratory has a rolling programme of recovery of toxoplasma from liquid nitrogen to maintain the routine system. In 28-9 the recovery success rate fell and there were consecutive failures. Initially with cultures stored from 4-1 years and subsequently with cultures stored for only 1-2 years. The storage and recovery procedures for the liquid nitrogen tanks were examined in detail. The most likely cause of the problem was a gradual change in tank maintenance which allowed the level of liquid nitrogen to drop below the level for effective preservation. The storage position in the tank then became critical as to whether samples were preserved. Despite the problems with liquid nitrogen recovery the toxoplasma culture system was robust enough to maintain the service. New SOPs are now in place. All stages of the process are now more strictly controlled and recovery rates have improved to 75%. Including one culture stored for 11 years and 7 months. Appendix C External QA Exception Reports 1. National Toxoplasma Reference Unit Serology 1 For the ISAGA IgM the Inverness result was negative ( /12) and the Swansea result positive (12/12). Repeat testing confirmed the Inverness results. 2. National Toxoplasma Reference Unit PCR Both laboratories reported inconsistent results for one sample containing 1 toxoplasma tachyzoites/2ul. This probably reflects sampling error and the limitations of the asasay. 12

13 Reference Laboratory Services Risk Log: May 29 Laboratory Risk Required action to mitigate risk Responsible Lead Deadline RAG Status Comment Scottish Toxoplasma Reference Laboratory Staff illness The laboratory has a small number of staff and has already experienced periods of extended sick- leave Integration within the Virology/Parasitology Department provides the staff support when required to maintain the service Dr DO Ho-Yen In place? Cell culture failure The laboratory relies on a continuous culture system to provide the toxoplasma tachyzoites for in- house tests. In particular for the dye test The laboratory has considerable stocks of cultures and cells in liquid nitrogen storage. There is a rolling recovery programme to ensure the quality of the stored material. Dr D O Ho- Yen In place? See update Appendix A 2.2 Information management system failure Used for results processing and reporting If this recovery system failed cultures could be obtained from the Toxoplasma Reference unit in Swansea to maintain the system The Medipath system is used throughout Microbiology and is supported by the IT department Dr D O Ho-Yen 13