Source document: https://docs-lawep.s3.us-east-2.amazonaws.com/1697633966017.pdf (Australia)
INVESTIGATION OF AUSTRALIAN BORRELIOSIS PATIENTS USING CURRENT TWO-TIERED SEROLOGY APPROACH Will Shirvington Tick-borne Diseases Unit, University of Sydney Supervisors: Dr Brent McParland, Dr Ann Mitrovic & Dr Carl Feng TICK-BORNE DISEASES UNIT Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 TBDU – University of Sydney › Much discussion regarding possible Borrelia spp.
in Australia › “Although locally-acquired Lyme disease cannot be ruled out, there is little evidence that it occurs in Australia.” – NSW Govt.
› Small number of doctors treating a growing number of patients › Research tick-borne illness in Australia › Current, major focus on Borrelia and Babesia Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Borrelia and Australia B. queenslandica isolated from rats First Australian case report of LB: Hunter Valley (Stewart et al.)

Russell et al.
process 12,000 ticks (NHMRC) 1962 1976 1982 1991 1994 1998 USA: Identification of B. burgdorferi causative agent of ‘Lyme disease’ Wills & Barry: ‘Borrelia-like spirochetes’ process 167 ticks NSW: Culture positive B. garinii Little, clear-cut evidence for presence of Borrelia in Australian ticks to support what Australian clinicians are seeing in their patients Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Diagnosing Borreliosis in Australia › Doctors not trained to recognise and treat the specific complexities presented by borreliosis › Lack of confidence in making clinical diagnosis of illness that is said to be absent in Australia › Reliance on diagnostic testing - CDC surveillance guidelines: two-tiered serology testing › First tier: ELISA ‘screening’ - If positive (or equivocal); › Second tier: Western blot Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Two-tier serology testing (CDC) › Lack of confidence making clinical diagnosis - over-reliance on serology › Assumes: - 1) Patient has functioning immune system (making antibodies) - 2) Free antibodies are present (not all tied in antibody/antigen complex) First Tier: ELISA - Combination of Borrelial antigens used - Detect antibodies against Borrelia If Positive/equivocal Second Tier: WESTERN BLOT - Only done when ELISA positive or equivocal - Positive western blot requires 5/10 Pos+ bands IgG 18 Osp C 28 30 Osp A Osp B 39 41 45 58 66 93 Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Limitations of ELISA Risk of excluding patients from treatment following negative ELISA › Only 55% (n = 45) of diagnosed patients returned positive ELISA, 100% returned positive western blot - Wojciechowska-Koszko et al., (2011) Serodiagnosis of borreliosis: indirect immunofluorescence assay, enzyme-linked immunosorbent assay and immunoblotting.
Archivum immunologiae et therapiae experimentalis 59(1):69-77 › Small number of antigens used in assay limits the possibility of the patient to return positive result › Institute for Clinical Pathology and Medical Research (ICPMR) - 2012: 1,935 samples tested w/ ELISA - 97 samples (5%) positive Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 › Important to tailor the criteria used to determine a positive western blot for the relevant population.
- Mavin S. et al., (2011).

Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

British journal of biomedical science 68(1): 5-10.
Western blot › ICPMR - Westmead - 280 samples positive/equivocal ELISA - 6 % returned positive western blot using CDC criteria › Considered that CDC criteria may not be effective for Australian experience and investigated › Mavin paper: investigates variety of WB criteria and finds most not well-suited to the Scottish experience › Criteria taken from Mavin et al., (2011) - Positive: 4 bands, weak positive: 3/15 18kDa 20 20 Osp C 28 43 30 Osp A Osp B 39 41 Osp B Osp A 43 48 45 48 58 66 93 Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Ongoing Study - Preliminary › Advertised through Karl McManus Foundation, Lyme Disease Association of Australia and treating clinicians 96 Diagnosed Borreliosis Patients 64 Healthy Controls FIRST TIER: ELISA SECOND TIER: Western blot ANALYSIS: CDC CRITERIA MAVIN CRITERIA Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 ELISA & Western blot used › ELISA - NovaLisa Borrelia burgdorferi IgG ELISA (NovaTec) - Not sure of species present in Australia - Recombinant epitopes in ELISA: - Osp C: B. burgdorferi s.s. & B. garinii - p100: B. afzelii - p18: B. afzelii - p41 (flagellin): B. garinii › Western blot - Euroimmun commercial IgG western blot - Whole cell lystates of B. burgdorferi, B. garinii & B. afzelii - Removed researcher bias using automation - BlotMaster - EUROLineScan software for evaluating each band Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 RESULTS - ELISA › CONTROL COHORT (n = 63) - 0 patients returned positive ELISA result › DIAGNOSED LYME COHORT (n = 96) - 3 patient returned positive ELISA result - 3.16 % › Suggests this ELISA kit not sensitive for Australian borreliosis patients › By current guidelines, 93 patients would be excluded from second-tier WB Growing evidence of an emerging
 tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 RESULTS – IgG Western blot Borrelia burgdorferi s.s. IgG DIAGNOSED LB (n = 96) CONTROL (n = 64) 9.38% 13.54% 22.92% 25.00% 52.08% 1.56% 1.56% 3.13% 37.50% p - value 0.052 0.009* 0.001* 0.091 59.38% 0.364 DIAGNOSED LB (N = 96) CONTROL (n = 64) p - value 1.04% 98.96% 0.00% 100.00% 1 1 Revised Criteria Positive Weak Pos Pos + Weak Pos Equivocal Negative CDC Positive Negative Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 RESULTS – IgG Western blot MAVIN POSITIVE Weak Pos Pos + Weak Pos Equivocal NEGATIVE CDC Positive Negative Borrelia garinii IgG DIAGNOSED LB (n = 95) CONTROL (n = 64) 5.26% 13.68% 18.95% 28.42% 52.63% 1.56% 3.13% 4.69% 28.13% 65.63% p - value 0.403 0.025* 0.009* 0.968 0.104 DIAGNOSED LB (N = 95) CONTROL (n = 64) 2.11% 97.89% 0.00% 100.00% 0.516 0.516 Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 RESULTS – IgG Western blot MAVIN POSITIVE Weak Pos Pos+Weak Pos Equivocal Negative CDC Positive Negative Borrelia afzelii IgG DIAGNOSED LB (N = 94) CONTROL (n = 64) 29.79% 27.66% 57.45% 29.79% 12.77% 14.06% 34.38% 48.44% 29.69% 21.88% p - value 0.022* 0.368 0.265 0.989 0.189 DIAGNOSED LB (N = 94) CONTROL (n = 64) p - value 7.45% 92.55% 3.13% 96.88% 0.313 0.313 Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 PRELIMINARY RESULTS • Positive western blot (Mavin criteria) responses: • B. afzelii: 54 • B. garinii: 18 • B. burgdorferi ss: 22 • Most common positive result: B. afzelii (32%) & B. garinii + B. afzelii (12%) RESULT MAVIN CRITERIA ( % ) (n = 94) CDC CRITERIA ( % ) (n = 94) p – value B. afzelii Positive Only B. burgdorferi Positive Only B. garinii Positive Only 2 SPECIES
 Positive 3 SPECIES Positive POSITIVE Negative 31.91% 9.57% 1.06% 19.15% 6.38% 68.09% 31.91% 5.32% 1.06% 1.06% 1.06% 0.00% 8.51% 91.49% Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Conclusion › 3 patients returned positive ELISA, however all 3 returned negative western blot result by CDC criteria.
- E.g by CDC criteria, 0 patients positive.

› Review the usefulness of ELISA as a ‘screening’ test in a two-tiered protocol.

› Look to alter western blot criteria to improve effectiveness.

› Isolating causal species is vital in creating a specific diagnostic test.

- Do we have the three species that were tested for?

- Is there a novel species that has antigens that are cross-reacting?
› In absence of reliable serology testing, clinical diagnosis based on the evolution of symptoms in a previously well patient following tick bite is essential.

› Educate and support Australian clinicians to be confident in diagnosing and treating borreliosis.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18 Acknowledgements › Participants › Supervisors: Dr Ann Mitrovic, Dr Brent McParland, Dr Carl Feng › USyd TBDU: Dr Mualla McManus, Ann Cincotta, Brooke Storey-Lewis, Mees Barak › Karl McManus Foundation › KMF - for organising the conference as well as the opportunity to present today TICK-BORNE DISEASES UNIT Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patientsSubmission 370 - Attachment 18