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Submission 545
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To the Senate :
Thank you for your invitation to provide a submission in relation to your Inquiry into the growing
evidence of an emerging tick-borne disease that causes a Lyme-like illness for many Australian
patients.
I write as the Director of Australian Biologics Testing Services, a private Pathology Laboratory
specialising in the detection of bacteria causing chronic illnesses. I wish to address Points A and C
and to a lesser extent Point D, as listed in the terms of reference for this Hearing.
Background :
Australian Biologics has operated as a laboratory since 1985. In 2002 our Molecular Biology section
was established. This involves the use of the Polymerase Chain Reaction (PCR) assay to identify the
presence of Borrelia DNA in human samples (blood, urine, tissue etc).
In August 2012 we introduced the Borrelia EliSpot LTT assay and in August 2013 we began offering
the Mikrogen IgM/IgG recomLine assay which measures IgG and IgM antibodies to antigens of
Borrelia. The EliSpot assays measures the amount of Interferon-gamma (a Cytokine) produced when
a patients Lymphocytes are exposed to antigens of Borrelia.
In response to Point A :
The prevalence and geographic distribution of Lyme-like illness in Australia :
Appendix 1 Geospatial Data (Pg. 11)
In response to Point C :
Laboratory Testing procedures for the detection of the Borrelia species in Australian Patients :
Limitations of Testing: There are multiple papers relating to the difficulties of accurate detection of
Borrelia. Testing by varying technologies is more likely to enable a definitive result of true positive
or true negative.
Serological testing has been shown not to be efficient for patients with a chronic infection of Borrelia
(see http://www.borreliose-gesellschaft.de/Texte/guidelines.pdf) as the infection causes
depression/exhaustion of the immune system which may cause false negatives. Unlike most other
bacterial infections, many patients do not sero-convert from IgM to IgG.
Earlier ELISA testing was known to have poor sensitivity whereas the newer ImmunoBlot assays
using recombinant antigens have a much higher level of sensitivity. The EliSpot Lymphocyte
Transformation Test is useful to show if an infection is active, although PCR testing remains the gold
standard for the detection of a bacterial infection.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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PCR is one of the most sensitive methods utilised to detect microbial pathogens in clinical
specimens. This is particularly necessary when specific pathogens, difficult to culture in vitro or are
known to be of low level in blood, tissue and other samples, are to be detected. The diagnostic
value of PCR is known to be significant.
To validate a PCR assay a laboratory enters into Quality Assurance Programme testing with an
accredited provider and/or obtains sequencing of the PCR product to confirm the validity of the
assay.
We have participated in QAPs for Borrelia with Quality Control Molecular Diagnostics (QCMD) based
at Glasgow University since 2014 .
Appendix 2.
Quality Assurance Programmes from QCMD for PCR testing for Borrelia. (Pg. 14)
Quality Assurance Programme from RCPA for Serology testing of Borrelia. (Pg. 22)
Appendix 4.
Appendix 5.
Appendix 6.
We utilise the following centres to sequence our PCR products - AGRF (the Australian Genomic
Research Facility) and the Ramaciotti Centre (a sequencing facility based at the University of New
South Wales).
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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test, it becomes possible to detect one specific cell out of 100,000 irrelevant cells, making this
method much more sensitive than traditional ELISA applications.
This method is an Interferon- assay for the specific detection of T-cells secreted due to a Borrelia
infection.
EliSpot (or ElisaSpot, short for Enzyme-linked Immunosorbent Spot Assay) was originally developed
as a method to detect antibody-secreting B-cells. Later the method was adapted to determine T-cell
reactions to a specific antigen, usually represented as the number of activated cells per million. Most
researchers use Interferon gamma (IFN-g) production as a read-out for activation of single cells.
Australian Biologics is a well-respected laboratory amongst doctors working with Borrelia infections
both in Australia and overseas. We regularly receive patient samples from clinics in Canada and
have tested samples from Asia, New Zealand and the United Kingdom. It appears that only in
Australia are we regularly subjected to slanderous attacks on our testing of Borrelia. Patients have
reported to us behaviour from senior Doctors and Specialists that involve quite outrageous
falsehoods about the quality of our testing.
For example, one specialist Practitioner told a patient who mentioned our testing, that he and some
colleagues had sent us water and that we returned a positive Borrelia result. We have been accused
of falsifying results in order to give the patient the result they want, patients have been told that
our laboratory is contaminated. The defamatory remarks are generally from Medical Specialists
none of whom have ever been to this laboratory or have had any dealings/discussions with us.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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Accreditation by NATA :
There are new regulatory requirements to be introduced for in-house IVDs. Under the new
regulatory framework for in vitro diagnostic medical devices (IVDs) that commenced on 1 July 2010,
laboratories manufacturing in-house IVDs will be required to meet the regulatory requirements to
legally supply their IVDs.
The Therapeutics Goods Agency is expected to introduce these new requirements for laboratory
inhouse testing in mid 2017.
Our PCR and recomLine assays are inhouse tests.
Inhouse tests are tests either developed from first principles; or developed or modified from a
published source; or used for a purpose, other than the intended purpose assigned by the
manufacturer.
To comply with the new legislation, we applied for NATA accreditation early in 2014. As there had
been no reason for our laboratory to go through accreditation (we do not seek Medicare payments
for our tests) we sought to have our Borrelia PCR assays accredited which includes accreditation of
the Laboratory.
This has not been an easy process and has not been aided by the disbelief of key figures at NATA
that we are detecting Borrelia. As mentioned earlier we have lodged with NATA copies of
sequences from patients tested and found positive for Borrelia by PCR. Our Quality Assurance
programmes with QCMD have been presented. Both sequencing and QAPs validate our PCR
procedures. As part of our validation we submitted a Probit Regression Study Appendix 7.
which entailed testing 850 samples and has shown our Borrelia PCR to be highly sensitive and with
good repeatability and reproducibility. We have shown correlation between our testing and other
overseas laboratories in our published research papers.
As NATA works jointly with the RCPA, the stance of the RCPA that no Borrelia exists in Australia
makes our accreditation problematic.
The International Standard for Laboratory Testing is ISO15189. Laboratories in all countries
undertaking accreditation must meet this standard. Due to the problems we have experienced with
NATA, we investigated the possibility of undertaking accreditation through an overseas accreditation
group.
Even though this is an international standard, in Australia no other accreditation groups will be
accepted. The TGA will not recognise any accreditation group apart from NATA.
There is enormous emphasis placed on NATA accredited laboratories compared to non NATA
accredited laboratories. It is necessary for an understanding of just what these differences mean in
reality.
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Submission 545
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Any laboratory wishing to claim Medicare rebates for their testing must have NATA accreditation.
This means the Government can be assured that the public money is going to laboratories that
comply with international standards, ISO 15189 and NPAAC requirements. Laboratories that do not
seek payment through Medicare have no legal requirement to go through NATA accreditation.
There are a number of small speciality laboratories operating in Australia who do not have
accreditation and have no need or intent to go through this process. Not having accreditation does
not mean that a laboratory is not giving accurate testing.
It is important to realise as well that University research laboratories do not have NATA
accreditation. We use two facilities for our sequencing of PCR products. One is the AGRF (Australian
Genomic Research Facility) which gained NATA accreditation a few years ago, the other is the
Ramaciotti Centre for Genomics at the New South Wales University which does not have NATA
accreditation. There is no suggestion made that results from the Ramaciotti cannot be trusted as
they do not have NATA accreditation.
The RCPA and Infectious Disease Society have relied on the research paper from Professor Peter
Irwin et al Bacterial Profiling Reveals Novel Ca. Neoehrlichia, Ehrlichia, and Anaplasma Species in
Australian Human-Biting Ticks. This paper was published December 28, 2015.
However earlier the same year, Professor Irwin published a paper Inhibition of the endosymbiont
Candidatus Midichloria mitochondrii during 16S rRNA gene profiling reveals potential pathogens in
Ixodes ticks from Australia published June 2015 in which he states the following :
Borrelia 16S sequences were obtained from ten questing I. ricinus ticks and a single I. holocyclus tick
removed from a wild Echidna (Tachyglossidae sp.). Borrelia sequences derived from the I. holocyclus
tick had 100 % sequence similarity, and clustered with high bootstrap confidence (91.1 %) into a
group of pathogenic relapsing fever Borrelia species including B. duttonii, B. recurrentis, B. parkeri
and B. crocidurae
Professor Irwins laboratory at Murdock University is not a NATA accredited laboratory.
The Dept. of Medical Entomology at Westmead Hospital does not have NATA accreditation.
Our Quality Assurance Programmes cannot be faked. The results prove that we have the ability to
detect Borrelia.
We participate in quality assurance programmes for both Molecular Biology and Serology, as we
strive to maintain high-quality standards in all areas of our work.
We are continuing our accreditation process despite the difficulties placed in our path.
D.
Evidence of investments in contemporary research into Australian pathogens specifically
acquired through the bite of a tick and including other potential vectors.
We have tested a large number of ticks and other potential carriers of the Borrelia bacteria. These
include bush lice and head lice. Borrelia DNA has been detected in all of these. Head lice (Pediculus
humanus capitis) was initially collected from a speciality hairdresser in a Sydney suburb not known
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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specifically as an endemic tick area. This collection of insects did not give a positive signal, however
at a later date, we were supplied with some Head Lice from a child who had tested positive for
Borrelia DNA. These Lice were found to be positive. We do not have the means to show
transmission however through such insects.
Samples from a horse drench were supplied to us for testing. The area of origin is known to us as
having positives in ticks from varying animals feral pigs, feral goats and horses. A strong positive
Borrelia DNA signal was found in eggs contained in the drench. We have not had these formally
identified but the horse had been infected with Botflys and the eggs may be from these Botflys.
Further research is needed to see how many animals may harbour the Borrelia bacteria.
Australian Biologics is part of a research group that includes Dr. Raphael Stricker of San Fransisco,
(Medical Director, Union Square Medical Associates, San Francisco, CA) Dr. Marianne Middelveen
(Microbiologist, Canada) and Professor Eva Sapi (Professor Biology New Haven University,
Connecticut). Studies generally utilise PCR for Borrelia from both Australian Biologics and New
Haven University.
Australian Biologics receives no remuneration from our research studies.
Appendix 8.
Evidence for Ixodes holocyclus as a vector for human Lyme borreliosis infection in Australia
http://www.ncbi.nlm.nih.gov/pubmed/25434042
Association of spirochetal infection with Morgellons disease.
http://www.ncbi.nlm.nih.gov/pubmed/24715950
Granulomatous hepatitis associated with chronic Borrelia burgdorferi infection: a case report
http://digitalcommons.newhaven.edu/biology-facpubs/33/
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In response to Submission 67s statement relating to our use of the Mikrogen recomLine
assay :
The paragraph below appeared in the submission from the anonymous Medical Specialist.
At no time have we been approached with any questions regarding our use of this test.
A laboratory scientist colleague recently worked in a laboratory in central Sydney that
specialises in diagnosing tick-borne infections. She reported that this laboratory was not
able to obtain NATA (National Association of Testing Authorities) accreditation due to their
poor scientific practices. She noted that if a patients antibody test was negative, the
scientist in charge would regularly tell them to incubate it for another day until it went
positive. This does not mean that the patient had a positive test rather it means the test
was performed incorrectly and the result is inaccurate. Antibody tests must be incubated for
exactly the duration specified by the test manufacturer, or results are not accurate. These
are the results that Australian patients are paying large amounts of money for, and are
holding onto as evidence they have diseases which they do not have.
Firstly I must say that we do not believe that any of our previous staff members would lie in
such a manner and the above charges are untrue. It would appear that the author of
Submission 67 has no real laboratory experience with ImmunoBlot testing as lengthening
the incubation time will not magically produce a positive result.
Secondly, it is not unusual for a laboratory to modify a kit test method. This makes the test
an in-house test which requires validation. We have performed the validation studies
required for this assay.
When we first began utilising Borrelia serology testing, we used a different assay to our
current Mikrogen kit. We changed to Mikrogen as we were not happy with the lack of
correlation between our serology testing and our PCR assays or between our serology and
results from overseas laboratories.
In mid 2014, I read a poster presentation titled Prolonged incubation and improved readout of the recomLine Borrelia burgdorferi IgM and IgG immunoblots (Mikrogen)
The following poster was presented at the 23rd meeting of the European Society of Clinical
Microbiology and Infectious Diseases by Doctors Goossens and Van Loo from the University
Hospital in Maastricht, the Netherlands.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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Serology: miscellaneous
Monday, April 29, 2013, 12:30 - 13:30
http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=163869&XNSPRACHE_ID=2&XNKONGRESS_ID=180&
XNMASKEN_ID=900
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Submission 545
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I spoke with Mikrogen about this and they suggested that increased incubation was not
required. We continued following the kit method for several months during which our
correlations with serology testing did not improve. I then managed to contact Dr. Van Loo
and spoke with her about this method. The conversation satisfied me that the method was
worth trialling so for the next few months we worked on validation of prolonged incubation.
Validation consisted of running all patient samples in tandem, one with 1 hour incubation
and a second with overnight incubation. We were able to confirm that overnight incubation
improved correlation with other more sensitive testing such as PCR and that the increased
incubation did not give false positives.
In relation to the comment made by Doctors Goossens and Van Loo Therefore, false
positive results have to be considered. Following is the statement that appears on our
result sheet for the recomLine assay.
Please note : An increase in ANA + EBV titres may induce false positives.
Practitioners are encouraged to test both ANA and EBV in patients with positive Blot results,
particularly with a positive IgM result.
Mikrogens production process is certified according to DIN EN ISO 13485. The Borrelia
recomLine IgM/IgG kit is CE-marked and matches the European Union IVD directive
98/79/EC.
Reading Submission 67, one would assume that all of our recomLine assays are sent out as
positive results. This is untrue. We find many more negative results than positive using the
recomLine kit. Statistics of patient results are found at;
Appendix 9. (Pg. 36)
A Quality Assurance Programme for Serological Testing of Borrelia species. This is a QAP
supplied by the Australian Royal College of Pathologists. This was passed successfully using
our prolonged incubation technique and with no false positives.
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Submission 545
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In response to Submission 362 relating to the accuracy of our testing
This anonymous Infectious Disease Specialist states the following :
I contacted the staff at Australian Biologics to ascertain how accurate their testing was. They sent
me data to say that testing on random samples of blood from patients presenting to a Rheumatology
Clinic and also random blood samples from well Australians showed positive results in approximately
70% and 35%, respectively. Thus the false positive rate appears to be extremely high with such
interferon gamma release assays. PCR testing by some of these groups uses short primers and a very
high number of cycles, both of which are known to be sources of false positives in all PCR tests.
Patients having these tests have often had a wide variety of investigations for their illness and are
understandably frustrated that their doctors cannot make a diagnosis easily. However, I am certainly
suspicious when they are charged over $1000 for a test with a very high false positive rate
The initial statement on interferon gamma release assays refers to the EliSpot LTT assay. This
statement is completely false. We have never carried out any testing on patients presenting to a
Rheumatology Clinic or performed random blood sampling on well Australians utilising EliSpot assays
or any other form of testing. If this doctor had called and asked for data on our accuracy of testing
we would have supplied our Quality Assurance Programmes from QCMD. The EliSpot assay is a kit
method and has been validated by the manufacturer AID Autoimmun Diagnostika GmbH in
Germany.
In relation to his comment on PCR, this statement is inaccurate.
To begin with, my staff are not allowed to divulge the origin and nature of reagents utilised in the
laboratory. With regard to our PCR technology, Australian Biologics uses probe mediated detection
technology which is by far the most specific PCR method for the detection of microorganisms. As a
result, false positivity can only occur by DNA contamination, however, we use the necessary controls
to know when false positives occur and runs are repeated to confirm positives when necessary.
That we have or would charge over $1000 for a test is also a falsehood. Our most expensive test is
the Borrelia EliSpot Lymphyocyte Transformation Test at $300, our PCR testing is $250 per sample.
Again it is very clear that this doctor has not had any contact at all with our laboratory and it is not at
all surprising that he has declined to provide his name.
Carl Sagan the late eminent astrophysicist stated Absence of Evidence is not Evidence of
Absence
We have provided evidence of presence that is being intentionally ignored.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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A
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
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APPENDIX 1B
SURVEYED
PATIENTS
Tested positive for
Borrelia; either never
left Australia or
developed symptoms
prior to travel.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
13 of 36
APPENDIX 1C
ALL PATIENTS
Tested positive for
Borrelia in Australia.
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX 2
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Individual Report
D at ase t code : 1
D at e Su bm it t e d : 19 D EC 12
Ms Jenni e Bur k e
Aus tr a l i a n Bi o l o g i cs Tes ti ng S er vi ces P /L
S yd ney - 2 0 0 0
Aus tr a l i a .
2 6 MAR 2 0 13
QCMD 2012 Borreli a burgdorf eri DNA EQA Programme - Indi vi dual Report
Tha nk yo u fo r p a r ti ci p a ti ng i n thi s Q CMD EQ A P r o g r a mme. Li s ted b el o w a r e the p a nel co d es a nd s a mp l e co ntents o f the
EQ A p a nel yo u r ecei ved .
Mat rix *
Copie s/m l
Bb D NA12 -0 3
Buffe r
B. b ur g do r fe r i D NA
10 0 ,0 0 0 c o p i e s/ml
Bb D NA12 -0 4
Buffe r
B. b ur g do r fe r i D NA
10 ,0 0 0 c o p i e s/ml
Bb D NA12 -0 2
Buffe r
B. afze l i i D NA
10 0 ,0 0 0 c o p i e s/ml
Bb D NA12 -0 5
Buffe r
B. afze l i i D NA
10 ,0 0 0 c o p i e s/ml
Bb D NA12 -0 1
Buffe r
Bo r r r e l i a sp p . D NA Ne g ati ve
Bb D NA12 -0 8
Cul tur e Me di um
B. b ur g do r fe r i
Bb D NA12 -10
Cul tur e Me di um
B. b ur g do r fe r i
10 ,0 0 0 c e l l s/ml
Bb D NA12 -0 6
Cul tur e Me di um
B. afze l i i
10 0 ,0 0 0 c e l l s/ml
10 ,0 0 0 c e l l s/ml
Bb D NA12 -0 7
Cul tur e Me di um
B. afze l i i
Bb D NA12 -0 9
Cul tur e Me di um
Bo r r e l i a sp p . Ne g ati ve
10 0 ,0 0 0 c e l l s/ml
D r An t on Van Loon
QCMD Exe cu t ive Co-ordin at or
QCMD 2012: The data and report documents provided are intended for the s ole us e of the participant. It is bas ed on material in our pos s es s ion or s upplied to us , which we believe to be reliable. Whils t every
effort has been made to ens ure its accuracy, we cannot offer any warranty that factual errors have not occurred. We therefore take no res pons ibility for any damage or los s that may be s uffered by reas on
of any s uch inaccuracies .
www.qcmd.org
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX152 of 36
20 12 Borrelia burgdorferi
An alysis of you r laborat ory's pe rf orm an ce on t h e core prof icie n cy sam ple s:
An alysis of you r laborat ory's pe rf orm an ce on all prof icie n cy sam ple s:
You r laborat ory's qu alit at ive re su lt s an d pe rf orm an ce score s
Qu alit at ive
Sam ple
Sam ple
St at u s
Sam ple
T ype
You r
qu alit at ive
re su lt
You r
qu alit at ive
score
Bb D NA12 -0 3
B. b ur g do r fe r i D NA
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA12 -0 4
B. b ur g do r fe r i D NA
D e te c te d
Co r e
p o si ti ve
Bb D NA12 -0 2
B. afze l i i D NA
D e te c te d
ne g ati ve
Bb D NA12 -0 5
B. afze l i i D NA
I nfr e q ue ntl y de te c te d
p o si ti ve
Bb D NA12 -0 1
Bo r r r e l i a sp p . D NA Ne g ati ve
Ne g ati ve
Co r e
ne g ati ve
Bb D NA12 -0 8
B. b ur g do r fe r i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA12 -10
B. b ur g do r fe r i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA12 -0 6
B. afze l i i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA12 -0 7
B. afze l i i
D e te c te d
p o si ti ve
Bb D NA12 -0 9
Bo r r e l i a sp p . Ne g ati ve
Ne g ati ve
Co r e
ne g ati ve
53
73.6 % o f a l l d a ta s ets
II
www.qcmd.o rg
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX 2
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Individual Report
D at ase t code : 1
D at e Su bm it t e d : 2 4 OCT 13
Ms Jenni e Bur k e
Aus tr a l i a n Bi o l o g i cs Tes ti ng S er vi ces P /L
S yd ney - 2 0 0 0
Aus tr a l i a .
2 3 DEC 2 0 13
QCMD 2013 Borreli a burgdorf eri DNA EQA Programme - Indi vi dual Report
Tha nk yo u fo r p a r ti ci p a ti ng i n thi s Q CMD EQ A P r o g r a mme. Li s ted b el o w a r e the p a nel co d es a nd s a mp l e co ntents o f the
EQ A p a nel yo u r ecei ved .
Mat rix *
ce lls/m l
Bb D NA13 -0 3
Cul tur e me di um
B. g ar i ni i
1.0 x10 E5
Bb D NA13 -0 5
Cul tur e me di um
B. g ar i ni i
1.0 x10 E4
Bb D NA13 -0 7
Cul tur e me di um
B. g ar i ni i
1.0 x10 E3
Bb D NA13 -0 8
Cul tur e me di um
B. b ur g do r fe r i s.s.
1.0 x10 E5
Bb D NA13 -10
Cul tur e me di um
B. b ur g do r fe r i s.s.
1.0 x10 E4
Bb D NA13 -0 2
Cul tur e me di um
B. b ur g do r fe r i s.s.
1.0 x10 E3
Bb D NA13 -0 4
Cul tur e me di um
B. afze l i i
1.0 x10 E5
Bb D NA13 -0 6
Cul tur e me di um
B. afze l i i
1.0 x10 E4
Bb D NA13 -0 1
Cul tur e me di um
T r e p o ne ma p hag e de ni s
1.0 x10 E5
Bb D NA13 -0 9
Cul tur e me di um
D r An t on Van Loon
QCMD Exe cu t ive Co-ordin at or
QCMD 2013: The data and report documents provided are intended for the s ole us e of the participant. It is bas ed on material in our pos s es s ion or s upplied to us , which we believe to be reliable. Whils t every
effort has been made to ens ure its accuracy, we cannot offer any warranty that factual errors have not occurred. We therefore take no res pons ibility for any damage or los s that may be s uffered by reas on
of any s uch inaccuracies .
1 of 2
www.qcmd.org
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX172 of 36
20 13 Borrelia burgdorferi
An alysis of you r laborat ory's pe rf orm an ce on t h e core prof icie n cy sam ple s:
An alysis of you r laborat ory's pe rf orm an ce on all prof icie n cy sam ple s:
You r laborat ory's qu alit at ive re su lt s an d pe rf orm an ce score s
Qu alit at ive
Sam ple
Sam ple
St at u s
Sam ple
T ype
You r
qu alit at ive
re su lt
You r
qu alit at ive
score
Bb D NA13 -0 3
B. g ar i ni i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA13 -0 5
B. g ar i ni i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA13 -0 7
B. g ar i ni i
Fr e q ue ntl y de te c te d
p o si ti ve
Bb D NA13 -0 8
B. b ur g do r fe r i s.s.
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA13 -10
B. b ur g do r fe r i s.s.
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA13 -0 2
B. b ur g do r fe r i s.s.
D e te c te d
p o si ti ve
Bb D NA13 -0 4
B. afze l i i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA13 -0 6
B. afze l i i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA13 -0 1
T r e p o ne ma p hag e de ni s
Ne g ati ve
Co r e
ne g ati ve
Bb D NA13 -0 9
Ne g ati ve
Co r e
ne g ati ve
60
6 5.0 % o f a l l d a ta s ets
2 of 2
www.qcmd.o rg
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX
18 2
of 36
Individual Report
D at ase t code : 1
D at e Su bm it t e d : 0 5 NOV 14
Ms Jenni e Bur k e
Aus tr a l i a n Bi o l o g i cs Tes ti ng S er vi ces P /L
S yd ney - 2 0 0 0
Aus tr a l i a .
19 DEC 2 0 14
QCMD 2014 Borreli a burgdorf eri s pp. DNA EQA Programme - Indi vi dual Report
Tha nk yo u fo r p a r ti ci p a ti ng i n thi s Q CMD EQ A P r o g r a mme. Li s ted b el o w a r e the p a nel co d es a nd s a mp l e co ntents o f the
EQ A p a nel yo u r ecei ved .
Mat rix *
Qu alit at ive
Ct valu e
Bb D NA14-0 1
BHI Br o th
Bo r r e l i a g ar i ni i
2 /2 Po s (10 0 % )
2 3 .5 2
Bb D NA14-0 7
BHI Br o th
Bo r r e l i a g ar i ni i
2 /2 Po s (10 0 % )
2 7.5 5
Bb D NA14-0 8
BHI Br o th
Bo r r e l i a g ar i ni i
2 /2 Po s (10 0 % )
3 1.9 2
Bb D NA14-0 9
BHI Br o th
Bo r r e l i a b ur g do r fe r i s.s.
2 /2 Po s (10 0 % )
2 9 .2 6
Bb D NA14-0 3
BHI Br o th
Bo r r e l i a b ur g do r fe r i s.s.
2 /2 Po s (10 0 % )
3 2 .9 3
Bb D NA14-0 4
BHI Br o th
Bo r r e l i a b ur g do r fe r i s.s.
2 /2 Po s (10 0 % )
3 5 .5 8
Bb D NA14-10
BHI Br o th
Bo r r e l i a afze l i i
2 /2 Po s (10 0 % )
2 5 .9 0
Bb D NA14-0 5
BHI Br o th
Bo r r e l i a afze l i i
2 /2 Po s (10 0 % )
2 9 .2 6
Bb D NA14-0 2
BHI Br o th
T r e p o ne ma p hag e de ni s
2 /2 Ne g (10 0 % )
Bb D NA14-0 6
BHI Br o th
Bo r r e l i a ne g ati ve
2 /2 Ne g (10 0 % )
D r An t on Van Loon
QCMD Exe cu t ive Co-ordin at or
QCMD 2014: The data and report documents provided are intended for the s ole us e of the participant. It is bas ed on material in our pos s es s ion or s upplied to us , which we believe to be reliable. Whils t every
effort has been made to ens ure its accuracy, we cannot offer any warranty that factual errors have not occurred. We therefore take no res pons ibility for any damage or los s that may be s uffered by reas on
of any s uch inaccuracies .
1 of 2
www.qcmd.org
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
19 of 36
APPENDIX 2
An alysis of you r laborat ory's pe rf orm an ce on t h e core prof icie n cy sam ple s:
An alysis of you r laborat ory's pe rf orm an ce on all prof icie n cy sam ple s:
You r laborat ory's qu alit at ive re su lt s an d pe rf orm an ce score s
Qu alit at ive
Sam ple
Sam ple
St at u s
Sam ple
T ype
You r
qu alit at ive
re su lt
You r
qu alit at ive
score
Bb D NA14-0 1
Bo r r e l i a g ar i ni i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA14-0 7
Bo r r e l i a g ar i ni i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA14-0 8
Bo r r e l i a g ar i ni i
D e te c te d
p o si ti ve
Bb D NA14-0 9
Bo r r e l i a b ur g do r fe r i s.s.
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA14-0 3
Bo r r e l i a b ur g do r fe r i s.s.
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA14-0 4
Bo r r e l i a b ur g do r fe r i s.s.
D e te c te d
p o si ti ve
Bb D NA14-10
Bo r r e l i a afze l i i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA14-0 5
Bo r r e l i a afze l i i
Fr e q ue ntl y de te c te d
Co r e
p o si ti ve
Bb D NA14-0 2
T r e p o ne ma p hag e de ni s
Ne g ati ve
Co r e
ne g ati ve
Bb D NA14-0 6
Bo r r e l i a ne g ati ve
Ne g ati ve
Co r e
ne g ati ve
T h e n u m be r of
Qu alit at ive dat ase t s
an alyse d
59
T h e su m of t h e
Qu alit at ive Pan e l Score
f or you r dat ase t is
2 of 2
8 9 .8 % o f a l l
d a ta s ets
www.qcmd.o rg
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
APPENDIX 2
Submission 545
Individual
Report
C at alo g ue C o d e :
QAB114147
Analysis T yp e :
Qualitative
D at ase t :
51533
20 of 36
R e p o rt UID :
3894/51533/267
Samp le C o nt e nt
Samp le St at us
BbDNA15- 01
Borrelia miyamotoi
BHI Broth
Negative
EDUCATIONAL
BbDNA15- 02
BHI Broth
Frequently Detected
CORE
BbDNA15- 03
BHI Broth
Frequently Detected
CORE
BbDNA15- 04
BHI Broth
Frequently Detected
CORE
BbDNA15- 05
BHI Broth
Frequently Detected
CORE
BbDNA15- 06
BHI Broth
Frequently Detected
CORE
BbDNA15- 07
Borrelia bavariensis
BHI Broth
Frequently Detected
CORE
BbDNA15- 08
Borrelia bavariensis
BHI Broth
Frequently Detected
CORE
BbDNA15- 09
Borrelia bavariensis
BHI Broth
Frequently Detected
CORE
BbDNA15- 10
Borrellia Negative
BHI Broth
Negative
CORE
[2]
[1] De t e ct ion Fre que ncy: To aid q ualitative analys is e ac h p ane l me mb e r is as s ig ne d a fre q ue nc y o f d e te c tio n. This is b as e d o n
the p e e r g ro up c o ns e ns us o f all q ualitative re s ults re turne d fro m p artic ip ants within the EQ A c halle ng e / d is trib utio n.
[2] Sample St at us: EQ A s amp le s are d e fine d as CO RE o r EDUCATIO NAL. Co re p ro fic ie nc y s amp le s are re vie we d b y the Q CMD
Sc ie ntific Exp e rt(s ). This is o n the b as is o f s c ie ntific info rmatio n, c linic al re le vanc e , c urre nt lite rature and , whe re ap p ro p riate ,
p ro fe s s io nal c linic al g uid e line s . Partic ip ating lab o rato rie s are e xp e c te d to re p o rt c o re p ro fic ie nc y s amp le s c o rre c tly within the
EQ A c halle ng e / d is trib utio n.
For further details please refer to the current participant manual.
N/A
[1]
Q C M D , Te c h n o l o g y Te rrac e , To d d C amp u s , We s t o f
Sc o tl an d Sc i e n c e Park , G l as g o w, G 20 0 XA Te l : +44 (0 ) 141
9 45 6 474, Fax: +44 (0 ) 141 9 45 579 5 We b : www.q c md .o rg
0
[3]
N/A
Pag e 1 o f 9
Is s u e D ate : 0 6 N o v 20 15
R e p o rt au th o ri s e d b y th e Q C M D Exe c u ti ve (1)
A U KAS ac c re d i te d p ro fi c i e n c y te s ti n g p ro vi d e r N o .438 5
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX 2
Individual
Report
C at alo g ue C o d e :
QAB114147
Samp le
Code
EQ A Asse ssme nt
G ro up C o nse nsus
BbDNA15- 01
SD
Analysis T yp e :
Qualitative
D at ase t :
51533
21 of 36
R e p o rt UID :
3894/51533/267
[5]
Yo ur
Q uant it at ive
R e sult [6 ]
Yo ur
Q ualit at ive
R e sult [7]
Positive
BbDNA15- 02
Positive
BbDNA15- 03
Positive
BbDNA15- 04
Positive
BbDNA15- 05
Positive
BbDNA15- 06
Positive
BbDNA15- 07
Positive
BbDNA15- 08
Positive
BbDNA15- 09
Positive
BbDNA15- 10
Negative
[4]
D e t e ct io n
Sco re [8]
Est imat io n
Sco re [9 ]
[1] EQ A Asse ssme nt Group: To aid d ata analys is , p artic ip ant re s ults are g ro up e d ac c o rd ing to the mo le c ular
amp lific atio n/d e te c tio n me tho d s p e c ifie d within the ir mo le c ular wo rkflo w fo r this c halle ng e / d is trib utio n. Fo r furthe r d e tails re fe r to
the Additional Information: Individual Panel Member Analysis s e c tio n o f this re p o rt.
[2] Core Pane l De t e ct ion (Q ualit at ive ) Score : An o ve rall c o re p ane l d e te c tio n s c o re p ro vid e d p e r c halle ng e / d is trib utio n.
[3] Core Pane l Est imat ion (Q uant it at ive ) Score : An o ve rall c o re p ane l e s timatio n s c o re p ro vid e d p e r c halle ng e / d is trib utio n.
[4] EQ A Asse ssme nt Group Conse nsus: The me an value fo r all re s ults within yo ur EQ A as s e s s me nt g ro up .
[5] SD: The s tand ard d e viatio n fo r re s ults fro m yo ur EQ A as s e s s me nt g ro up .
[6 ] Your Q uant it at ive Re sult : The q uantitative re s ult yo u re turne d fo r e ac h s amp le within this EQ A c halle ng e . LO D/NR (limit o f
d e te c tio n o r no t re p o rte d ).
[7] Your Q ualit at ive Re sult : The q ualitative re s ult yo u re p o rte d fo r e ac h s amp le within this EQ A c halle ng e / d is trib utio n.
[8 ] De t e ct ion Score : Yo ur d e te c tio n (q ualitative ) s c o re s are b as e d o n the as s ig ne d d e te c tio n fre q ue nc y o f e ac h p ane l me mb e rs ,
whe re 0 (z e ro ) is hig hly s atis fac to ry and 3 (thre e ) is hig hly uns atis fac to ry. Sc o re s are p ro vid e d fo r ind ivid ual p ane l me mb e rs .
[9 ] Est imat ion Score : Yo ur e s timatio n (q uantitative ) s c o re s are c alc ulate d b as e d o n yo ur variatio n fro m the c o ns e ns us fo r yo ur EQ A
as s e s s me nt g ro up . With 0 (z e ro ) s c o re d if the q uantitative value yo u re p o rte d is within o ne s tand ard d e viatio n (SD) fro m yo ur EQ A
as s e s s me nt g ro up c o ns e ns us , 1 (o ne ) if yo ur q uantitative value is b e twe e n o ne and two SDs , 2 (two ) if yo ur q uantitative value is
within two and thre e SDs and 3 (thre e ) if yo ur q uantitative value is mo re than thre e SDs fro m the me an o f yo ur EQ A as s e s s me nt
g ro up .
For further details please refer to the current participant manual.
Q C M D , Te c h n o l o g y Te rrac e , To d d C amp u s , We s t o f
Sc o tl an d Sc i e n c e Park , G l as g o w, G 20 0 XA Te l : +44 (0 ) 141
9 45 6 474, Fax: +44 (0 ) 141 9 45 579 5 We b : www.q c md .o rg
Pag e 2 o f 9
Is s u e D ate : 0 6 N o v 20 15
R e p o rt au th o ri s e d b y th e Q C M D Exe c u ti ve (1)
A U KAS ac c re d i te d p ro fi c i e n c y te s ti n g p ro vi d e r N o .438 5
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
APPENDIX 3
RCPA Quality
Phone
Assurance Programs
Fax
ABN
+61 2 9356
22
of 2003
36
32 003 520 072
www.rcpaqap.com.au
Survey Report
Participant Number 1074
Copyright
This material is copyright and may n
any purpose whatsoever (inclu
Limited. Permission must be sought in writing from the Program but will not be unreasonably refused.
Confidentiality
RCPA Quality Assurance Programs Pty L
party, unless required by legis
papers to journals.
2015 RCPA Quality Assurance Programs Pty Ltd. All rights reserved
File
S4:2015
Report Issued
Page
Page 1 of 11
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APPENDIX 3
23 of 36
Survey Report
2015 RCPAQAP Serology. All rights reserved
RCPA Quality Assurance Programs Pty Ltd keeps all participant details confidential. Such details
will not be disclosed to a third party, unless required by legislation, without the prior written
consent of the participant.
Parasite & Spirochaete Module Report for Survey S4:2015 Due Date: 23 Oct 2015
Participants: 14
Test
Lyme Disease IgG
Spec
4E
Consensus
pos
Result
100%
4E
neg
4E
No Consensus
4E
4E
Graphs
Your Result
NA
Your Score
-
Kit in date
-
Total Score
-
Possible
-
100%
NA
NA
No Consensus
pos
No Consensus
neg
Total Score:
Test
Lyme Disease IgG
Spec
4F
Consensus
No Consensus
Result
-
4F
pos
4F
No Consensus
4F
4F
Graphs
100%
Your Result
NA
Your Score
-
Kit in date
-
Total Score
-
Possible
-
100%
NA
NA
No Consensus
pos
No Consensus
pos
Total Score:
Accredited for compliance with ISO/IEC 17043
Accreditation Number: 14863
Page 2 of 11
RCPAQAP Serology
Suite 201, Level 2
8 Herbert Street
ST LEONARDS NSW 2065
Phone: +61 2 9045 6070
Fax: 1300 78 29 21 (Aus)
+ 61 2 9356 2003 (Intl)
ABN: 32 003 520 072
100%
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Submission 545
APPENDIX
24 of 36
Survey Report
2015 RCPAQAP Serology. All rights reserved
Cumulative Year to Date Scores for Participant 1074
Survey
Your Score
Possible Score
Your Percent
All Participants
S4:2015
100%
94%
100%
Page 4 of 11
RCPAQAP Serology
Suite 201, Level 2
8 Herbert Street
ST LEONARDS NSW 2065
Phone: +61 2 9045 6070
Fax: 1300 78 29 21 (Aus)
+ 61 2 9356 2003 (Intl)
ABN: 32 003 520 072
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
25 of 36
Appendix 4. Sequences
Lab
number
sample
Date
CM0906
Tick
29/10/14
CO1852(a)
CP0271
CO0676
CO1995
Tissue
(Site of Bite)
Blood
Serum
CSF
30/10/15
24/11/15
24/11/15
24/11/15
Organism
Borrelia garinii
Identity
98%
Borrelia burgdorferi
Borrelia burgdorferi
Borrelia burgdorferi
Borrelia burgdorferi
99%
100%
99%
99%
128
107
134
141
Sequences
>CM906
TATATTCCAGCCGGTaAGCATCTTTTGGTTAGAGATGGAGATGTTGTTAAAGCAGGCGATATGCTTTGTGATGGCAGA
ATTAATCCTCATGATGTGCTTGAAATTTTAGGTGGGA
NCBI Blast: https://drive.google.com/file/d/0By5_nsCaD9lob2xRdXN5Z2djLWM/view?usp=sharing
> CO1852(a)
CCGTTGGCAATCTACCATCACAAGCATATCTCCTGCTTTTACAACATCTCCAGTCTCTAACCAAAAGATGTTTTCCAGCT
GGAATATAATGCTTATGTTCAACCCCATACTCATCTAAAATATTAATAAGCCTTTTACCTTTTTGAATTGA
NCBI Blast: https://drive.google.com/file/d/0By5_nsCaD9loQ2lhdkhXM3pVTHc/view?usp=sharing
>CP271
GACTAGTGAACTGCATTCAAGCCTATCTCCTTTTTTGATTGAAACACATCTCCATCTCTAACCAAAAGATGTTTTCCAGCT
GGAATATAATGCTTATGTTCAACCCCATACTCATCTAAAATATTAATAAGCCTTTTACCTTTTTGAATTGAA
NCBI Blast: https://drive.google.com/file/d/0By5_nsCaD9loeWR6YkhGb05wRjQ/view?usp=sharing
>CO676
GCTAGTACTCTACATCACAAGCATATCTCCTGCTTTTACAACATCTCCATCTCTAACCAAAAGATGTTTTCCAGCTGGAA
TATAATGCTTATGTTCAACCCCATACTCATCTAAAATATTAATAAGCCTTTTACCTTTTTGAATTGAATT
NCBI Blast: https://drive.google.com/file/d/0By5_nsCaD9loUmRsYUhTWFh5XzA/view?usp=sharing
>CO1995
AATAGTCTCTACATCACAAGCATATCTCCTGCTTTTACAACATCTCCATCTCTAACCAAAAGATGTTTTCCAGCTGGAAT
ATAATGCTTATGTTCAACCCCATACTCATCTAAAATATTAATAAGCCTTTTACCTTTTTGAATTGAA
NCBI Blast: https://drive.google.com/file/d/0By5_nsCaD9loSnNZU2Joc1laLXM/view?usp=sharing
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A
l
P
&
&
&
Borrelia
Elispot
Immunoblot
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Submission 545
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0
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Page #:
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APPENDIX 7
2: False positives
To assess the false positive rate, a total of 84 reactions were performed on samples from two healthy donors.
A fluorescence threshold of 0.03 R was established such that the background did not interfere with our readings, especially
at high cycle numbers (>40). As Positive control were used 10 copies of B.burgdorferi; as negative control we used distilled
DNAase, RNAase-free water.
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APPENDIX 7
Page #:
2 of 2
Results:
1. DNA extraction validation
In order to evaluate the efficiency of our DNA extraction method we compared a standard curve obtained diluting the
B.burgdorferi control DNA in TE buffer (Fig 1a) with a standard curve obtained after spiking control DNA into different
sample types (Blood Concentrate; Serum; Urine; CSF and Tissue) and performing DNA extraction (Fig 1b). A detailed
explanation of the spiking protocol is provided in Appendix 1.1 (serial dilution 1).
Ct value
40
y = -3.35x + 36.7
35
B.burg
30
25
20
-1
log(copies)
(a)
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APPENDIX 7
Page #:
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Ct value
40
y = -3.3x + 36.7
Urine
35
Serum
Tissue
30
CSF
average
25
-1
Linear (average )
log(copies)
(b)
Figure 1: (a) standard curve before extraction; (b) after spiking in different samples and performing DNA extraction.
From the equations of the standard curves we calculate PCR efficiencies of nearly 100% both before and after extraction.
Similarly, the intercepts (i.e.: the Ct value for one copy of target DNA) were 36.7 cycles both before and after extraction,
which confirms that our DNA extraction method is reliable and quantitative (Appendix 1.3.)
2. False positive rate
In order to assess the false positive rate of the Borrelia RT-PCR assay we performed 84 reactions using negative samples
from 2 healthy donors and we observed no positives using a threshold of 0.03 R. Using a Bayesian approach (Gelman et
al. 2004; Carlin and Louis 1996, Appendix 2) we estimated the posterior mean false positive rate as (0+1)/(84+2) = 1.1%,
with one sided 95% credible interval of [0, 3.5%]. This places an upper limit of 3.5% for the false positive rate for a single
reaction. However, it is important remind the reader that it is our standard practice to process water samples along patients
samples in order to control for contamination during the DNA extraction or the PCR setup steps. Moreover, by performing
the PCR reactions up to 6 times, we reduce the probability of false positive calling. See section 4 for false positive
probability calculations.
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APPENDIX 7
Page #:
4 of 2
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Rev.:
Laboratory Manual
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APPENDIX 7
0
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Page #:
5 of 2
Figure 3: probit curves for individual sample types. Colour coding as per legend. Triangles: observed data points; dashed lines: probit
regression predictions.
Given the fact that the probability of detection is not affected by the sample type, we can provide a general table of
detection probabilities depending on DNA concentration (Table 3.4).
bacteria
Conc (copies/ml)
log.conc
P(Detection)
B.burgdorferi
40000
10.60
1.000
B.burgdorferi
8000
8.99
1.000
B.burgdorferi
1600
7.38
1.000
B.burgdorferi
320
5.77
1.000
B.burgdorferi
64
4.16
0.996
B.burgdorferi
32
3.47
0.956
B.burgdorferi
16
2.77
0.775
B.burgdorferi
12.8
2.55
0.673
2.08
0.422
B.burgdorferi
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APPENDIX 7
Page #:
6 of 2
1/6 = negative;
2/6 = equivocal;
3/6, 4/6 = positive.
Given the probabilities of detection predicted by our probit model (Table 3.4) and applying the above-mentioned rules we
calculate that at 16 copies/ml we achieve a probability of detection of 97.3%, with a false negative rate of 1.3%.
Despite the 0% found in the 84 tests for a false positive, the Bayesian analysis in section 2 allowed false positive rate
estimates to be made as 3.5% (at the 95% probability level).
Using the same criterion for a true positive as given above, the false positive rate is 1.3%.
(For probability calculations, see Appendices 4.1, 4.2 and 4.3).
5 : Limit of detection
In order to ensure a probability of detection greater than 77%, as predicted in the probit regression model, we set our limit
of detection as 16 copies/ml (See Table 3.4). However, we successfully detected amplification signals from as low as 8
copies/ml: this detection was confirmed by end-point amplification and sequencing (Appendix 5).
6: Repeatability
Our data were analysed to assess the consistency of results obtained by the same operator across different tests (i.e.:
repeatability). As a unit of measure we used the following: % Repeatability = 100 - % Standard Deviation.
(5 repeats per measurement). Appendix 6 shows the data from repeatability studies.
Since we want to exclude the variability of the assay due to low template concentrations, we focussed on the data obtained
from samples with a concentration greater than 64 copies/ml. As shown in Figure 5, the repeatability of results by each
operator across all sample types and concentrations was above 95%.
% repeatability
Repeatability
100%
99%
98%
97%
96%
95%
94%
93%
92%
91%
90%
8,000
1600
320
Concentration (copies/ml)
64
Tissue (Maira)
Tissue (Yean)
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APPENDIX 7
Page #:
7 of 2
Fig 5: repeatability of tests: percentage repeatability of results across different DNA concentrations and different sample types. (a) B.Burgdorferi data;
7: Reproducibility
The reproducibility of results performed by two operators (ie: reproducibility), was assessed comparing the means of each
data point and performing a Students t-test for paired samples: applying a Bonferroni adjustment for multiple testing, we
observed no statistical difference in 36 pairwise comparisons (T-test p values in Appendix 7). All the P-values are above
the adjusted significance level, which means that there is no statistically significant difference between the measurements of
different operators.
40,000
8,000
1600
320
64
0.94
0.42
0.73
0.95
0.80
0.49
0.04
0.35
0.91
0.87
0.31
0.67
0.40
0.38
0.39
0.44
0.28
0.11
0.77
0.94
0.93
0.08
0.21
0.09
0.57
64
32
16
0.72
0.23
0.02
0.05
0.46
0.30
0.56
0.22
0.82
Unless signed by author, authoriser and/or approver, printed copy is controlled document only on date last printed 08/04/2014
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
Rev.:
Laboratory Manual
0
35 of 36
APPENDIX 7
Page #:
8 of 2
References
Gelman, A., Carlin, J.B., Stern, H.S. and Rubin, D.B., Bayesian Data Analysis, 2004, 2nd Ed., Boca Raton, FL , Chapman
& Hall/CRC
Carlin, B.P. & Louis, T.A. (1996), Bayes and Empirical Bayes Methods for Data Analysis, Chapman & Hall, London
Approved by
Date
Unless signed by author, authoriser and/or approver, printed copy is controlled document only on date last printed 08/04/2014
Growing evidence of an emerging tick-borne disease that causes a Lyme like illness for many Australian patients
Submission 545
36 of 36
A