DAKO / Oxoid Borrelia antibody test kit brochures, with quotes and critical comments by Marie Kroun, MD:

LATEST NEWS May 2, 2010, INFORMATION.DK (in danish, PDF print)
Klaus Hansen - one of the DAKO/OXOID test inventors (co-work with Lebech, does she also earn money on the test?) - has admitted that he earns royalty money on every sale of the OXOID antibody test kits!
Translation of article into English and comment http://lymerick.net/Hansen-conflict.html
Despite scientific journals have required authors to declare eventual conflicting interests for a long time in their publications, Klaus Hansen "forgot" to declare his income on his flagella antibody test; he has been the main advisor on Borrelia test in Denmark since 1985!

In spring 2009, I made Statens serum institut (SSI) - the danish state national microbiology reference laboratory - aware of the probable conflict of interest of Hansen and Lebech in their articles; but that "reminder" did not lead to the declaration of Hansens conflict of interest, a year ago - not until the press was put on the fault!

I asked SSI a lot of questions on the test, was told "it was developed in-house on SSI" and the price SSI sell it for.
I got a reference list of 19 articles, which all had Hansen and Lebech as co-authors, showing that SSI rely their statements on these authors work, solely!

I asked for information about ownership, patent or trademark number, I did not get that information from SSI! - why not?

I asked for quality control data - which SSI as danish national reference laboratory should be able to provide - i.e. measures of intra- and inter-laboratory variation of test results done on the same control sera; since testing controls are mandatory according to manufacturers specifications and required in every single test, because the OD of the patient sample is compared to OD of known positive and negative control sera! -  I could not get that information either!
Tthus, either SSI does not save their control measures (i.e. they lack controlling, CLIA style!?)  or SSI dare not reveal their control measure data in public, probably because there is so much variation in repeated measures of test results, that it would display how uncertain results of this test really are?! - as it has been shown for other serology tests! 

Information about the danish flagella antibody test come from 3 kit insert / manufacturers specifications, that is apparently not publicly available on Internet:
Worth noting also is that the clinical selection of "typical" neuroborreliosis AKA Bannwarth syndrom favors infections caused by B. garinii, but others strains of Borrelia burgdorferi sensu lato may differ from the above mentioned expected test "performance" and also clinically! - so the above "expected performance" may only be true for B. garinii neuro-infection?

In my experience from late Borrelia patients many have been diagnosed with ME/CFS or fibromyalgia or psychosomatics, without having been properly evaluated for medical explanations of "chronic fatigue"; many have not been properly evaluated for chronic (tickborne) infections, even despite they have had more tickbites in past history and may even have had a "previous" borrelia diagnosis, thought by doctors, but usually not the patient who continue to ask for better tests and treatment for suspected borreliois and other tickborne infections ...  they state Borrelia infection must have been cured effectively by conventional 10-14 days antibiotic treatment (how about us who get 5-10 new tickbites every year and get new EMs?) ... the above patient have relapsed many times, Borrelia antigen (exact species unknown) was proven by direct fluorescent antibody stain for Borrelia burgdorferi in 2001 and spirochaetes was seen in blood twice at 8 months interval in 2008.
Many more cases like it - see persistent-borreliosis reference list - in which I focus on culture, microscopy and/or PCR confirmed cases of either late diagnosed untreated and/or persistent Borrelia infection after conventional treatment for Borrelia infection.

Out of 12/33 patients participating in my pilot-study (see my York 2003 PowerPoint presentation; patients overview) were diagnosed with ME/CFS before entering my Borrelia antigen test project.
All 33 patients self suspected they might have persistent Borrelia, because of known previous tickbite(s) and/or EM(s) and/or perhaps a positive Borrelia serology at some point in past history and not the least because of RECURRENT FLARE ACTIVITY, consistent with Borrelia growth pattern. All 33 patients were tested during CURRENT DISEASE ACTIVITY i.e. when expressing cyclical relapse pattern, either weekly or monthly, sample preferably taking during the first day of a new flare, because in old published words on relapsing fever Borrelia, spirochetes were only visible in the blood during the active disease days (usually accompagnied by febrilia which is usually not the case in Lyme borreliosis), but the blood was still contagious in between flares, when there were no visible spirochetes in the blood! - making the logical reasoning that if we want to find spirochetes in Borreliosis, we need to time sampling to the day Borrelia enter blood from tissues in its spirochete form, and create a fierce immunreaction i.e. symptoms!
All 33 patients who were tested (many more were interested in participating in my project, but did not have current relapse pattern and were not tested until they eventually went into overt relapse) had Borrelia antigen in their blood stained positive by direct immunofluorescent stain for Borrelia burgdorferi! ...
Further 17 patients were tested in the long-term follow-up project and also tested positive on direct immunofluorescent stain for Borrelia; unfortunately in spring 2007, I had to stop enrolling more patients into the intended to be long-term follow-up project, because my intention was to take a 5-year after enrolling / after positive direct fluorescent antibody test for Borrelia burgdorferi  - in the hope to see some patients had been cured and came out negative on direct fluorescent antibody test results; however the USA laboratory decided to change the Borrelia antigen test from detection by microscopy, to instead counting the of number of fluorescent structures by computer (flowcytometri); thus, it would never be possible for us to get any post-treatment sample done with the same method, as when the patients enrolled in the study; besides the lab. also stopped doing simple microscopy of blood-smears to look for other tickborne co-infections - and I got no pictures back to compare my own microscopy result on fresh sample to ...
- so in future project we will instead will try to culture for Borrelia and if success do direct fluorescent antibody stain (if acridin orange is positive for DNA/RNA), and if money permits PCR, plus of course compare culture positive with result of the danish serology test and a LineBlot method ... 
It is noteworthy that - according to the danish definition / diagnostic criteria for ME/CFS - it is a necessity to look for and outrule all other more common causes of chronic fatigue: malignancy, autoimmunity, chronic bacterial or parasitic infections, neuromuscular, endocrine (esp.hypothyroid and hypo-adrenal gland function), hematological and internal medical diseases, psychiatric disorders, drug abuse and toxin exposure 
- all this must be properly investigated for all with negative results, but testing for all these conditions are often NOT DONE, probably for economic reasons?! - many times patient is doomed psychosomatic or hypocondriac even without being seen by a psychiatrist!
Thus, 3/12 (25%) of the pilot-patients, who were formerly diagnosed with ME/CFS, had never been tested for Borrelia antibodies (despite tick-bite history preceding development of chronic fatigue!).
NONE of the 33 pilo-project participants had been tested for any of the other possible tickborne infections (anaplasma, ehrlichia, rickettsia, babesia, bartonella, TBE ... plus more other), that may infect the patient from a tickbite alone or may coexist together with Borrelia in the patients, who developed their chronic illness months to years after tickbite! ...
75% of 33 pilot-project participants had microscopic sign in (from 2002 mainly buffy-coat was examined) blood smears on hitherto uninvestigated and therefore undiagnosed co-infections that could be spotted on simple stained blood smear!
All enrolled patients had been through the normal danish medical evaluation system, who had given up and told the patient "you just have to learn to live with it" i.e. any treatment success would be better than the patient could have hoped for. Most of the treated improved a lot (> 50%, usually 75% for long term sick >2 years and 100% for those sick less than 2 years) on antibiotic treatment directed to hit all found infections, despite we only had access to treat with oral drugs; but unfortunately the danish Drug Administration (Lægemiddelstyrelsen) in 2002 denied me permission to import atovaquone for treating project participants diagnosed with ringforms in their red blood cells; due to commen serious and perhaps permanent  adverse  effects  of quinine, this drug was not attractive to use in low-level (0,1-1% parasitaemia) infected patients  ... so low infection level is below what gives overt babesiosis symptoms, blood piss and anaemia; thus many with this co-infection were not treated for it at all, some took Malarone, I personally got atovaquone suspension (legal to import for own use if one can get a prescription) improved a lot on it, but not lasting - could have been easily reinfected the following month, where I got 4 new tickbites, after which symptoms came back  ... ringformed parasites could still be detected after more treatment courses!
Microhematokrit technique - or using a newer term Quantitative Buffy-Coat (QBC) - has become the international standard for diagnosing malaria in endemic countries, because Malaria infection can be detected in pre-symptomatic stage and treated with high chance of cure before sickness develop. For a year I compared peripheral ear prick samples with buffy-coat smears and found the latter much more sensitive and quicker microscopy to first parasite detection. That part of my study was also controlled by italian vet. Walter Tarello, who did not know any of the patients histories or laboratory findings; we could both find all positives the US lab. had found in simultaneously taken smears, and found more, probably because we did microscopy for 3-4 hours per patient where the US lab. only could use 30-60 minutes per patient because of higher work load! - meticulous search of two large smears paid off ...   
Coinfections complicate both the clinical picture, the response to antibiotic treatment and the test results, since some of them infect and kills immune cells, so immunological / serological reactions may not be as usually expected in patients with Borrelia (garinii?) infection only.

UNFORTUNATELY - despite the warnings in the test kits about improper use of serology test results - many danish microbiologists and clinical doctors improperly use a positive Borrelia antibody test to conclude that Borrelia burgdorferi must be the most likely cause of the patients symptoms and use positive serology test result to offer the serum Borrelia antibody test positive patient antibiotic treatment
- while they on the other hand usually totally exclude the possibility of the patient having a currently active Borrelia burgdorferi infection, whenever the Borrelia serology test comes out negative!
The doctors have not been informed from the health authorities and have not self read the relevant literature, and are simply not aware that positive SEROLOGY test results does not reflect Borrelia disease activity, rather the opposite!
When serology is positive the patient is usually in a better condition, compared to when serology is negative but spirochaetes detectable by simple microscopy; the latter patient may be very, very sick and in dire need of testing and treatment, but is offered nothing because of negative serology is used to outrule Borrelia infection with 100% certainty, if the patient has been sick over 3 months :(
Antibiotics helps patients with spirochaetes in blood detectable by microscopy, but it can be a rough start, due to Jarisch Herxheimer reaction, due to killing many spirochaetes at once!
- but because of Borrelia's ability to go into alternative resistant forms, in in such a state may survive adverse conditions for years, for later if/when the environment again give the microbes favorable growth conditions, some patients will relapse, but can usually again get help from renewed antibiotic treatment.  There is no good reason to deny patients treatment that the patients feel helps; in my experience patients do not continue ineffective treatment, because of cost, side effect etc.!  

The OXOID / DAKO test kit brochures does not specifically mention that in case lots of Borrelia ANTIGEN is present, some or all the formed ANTIBODY may immediately be bound to antigen in IMMUNE-COMPLEXES, that mostly will precipitate inside the patient (inducing vasculitis? ... perivascular inflammation with chronic inflammatory cells are the micro-pathologic hallmark of Borrelia infection, and many symptoms can be easily explained by reduced microvascular circulation in the tissues; decreased brain perfusion in ares corresponding to the patients "functional complaints" can be visualized by SPECT and similar methods - but is available in DK for routine testing!), hence a situation can arise, that there is perhaps NO SURPLUS OF FREE UNBOUND ANTIBODIES left in circulation, that may bind to the TEST ANTIGEN, hence the test will measure NOTHING - the result is a FALSE NEGATIVE SEROLOGY TEST OUTCOME, no matter which test is used, but of course related to chosen cut-off level for positive test. 

It is wellknown from the literature that symptoms of persistent Lyme borreliosis usually will fluctuate much over time, just like relapsing fever; sometimes patients are only symptomatic for a few days in a row, sometimes for some weeks in a row and rarely for several months in a row
- unless there are co-infections or other immune depressing conditions also involved in the persistent disease named "chronic / persistent Lyme disease"
- which is probably often the case in persistent Borrelia that flare more and longer when the patient is immune depressed!
- but when longer relapses recur again and again, and especially during periods with relapses coming at short interval of 8-10 days, where the patients are totally invalidated (often severe migraine like headache) for 2-3 days and moderately ill all the other days, working become practically impossible; patients need to take long-term sickleaves and often will loose their job after a year.
After that economic deroute is added to the health deroute, and people may not be able to pay for testing and treatment, when it is not offered for free by the danish hospital system. This hits the whole family and patients feel life is not worth living anymore, when they are unable to contribute to the family economy, and in pain all the time and no future improvement ahead - suicide has been the result in at least one case I know, and many have been very close to ...
- since they are denied all other tests than the abovementioned serology test that often fail in the most ill.  WE SHOULD BE ABLE TO DO IT MUCH BETTER!

The symptomatic episodes were already in 1979 shown to be associated with presence of circulating IgG immunecomplexes by Hardin, Steere et al. PDF:
"The presence of reactive material correlated with clinical aspects of disease activity; it was found early in the illness, was most prominent in sera from the sickest patients, was infrequent during remissions, and often fluctuated in parallel with changes in clinical status." ... "Furthermore, the seasonal onset and cyclical nature of the illness and the presence of a pre-articular phase (especially ECM) would make it a convenient human model for studying how early immune responses are related to the development of inflammatory joint disease and other clinical events. Consequently, we have looked for evidence of circulating immune complexes in the serum of patients with Lyme arthritis by three standard assay systems ..."

Just a few years later Borrelia burgdorferi was found (Science 1982, PMID: 9673306) to be the cause of Lyme arthritis / Lyme disease / Lyme borreliosis, and just a few years later more EU Borrelia strains - Borrelia burgdorferi sensu strictu, Borrelia garinii and Borrelia afzelii - were found associated with Borrelia - described in Europa for about a century! - and early was noticed differences in the Lyme disease illness expression in B. garinii and B. afzelii infections compared to Borrelia burgdorferi sensu strictu - broadening the clinical spectrum of Lyme borreliosis.

Within the last decennial 3 more Borrelia strains have been detected in sick EU people: Borrelia lusitania, Borrelia valaisiana, Borrelia spielmanii (ALL SERONEGATIVE) - plus other strains in other parts of the world ... plus many other Borrelia strains (counting hundreds) have still only been detected in ticks so far, but they probably have a tick-mammal cycle also, we must suppose, since that is what Borrelia usually does? - but all these strains have not been detected in sick humans yet, can and do any / all of these cause illness in humans? - that is the question we still have no answer to yet ...

Borrelia burgdorferi sensu lato was detected in preserved museum specimens from late 1800, i.e. at about the time when the atrophic stage of ACA was first described in the medical literature (see my Hull 2001 Borrelia history lecture, or the German version from 2006 Borrelia history lecture in Kassel) - so Borrelia burgdorferi sensu lato is certainly not a NEW microbe; so the newest of the "new" strains have probably been around for a long time too, maybe just as long as the others, but they differ so much in their surface antigens and genes, from the three old EU Borrelia variants that commonly used SEROLOGY tests fails to detect them.
The "new" strains were only found after Borrelia genome and strains differences were found and genetic testing with PCR test became available in some counties, since only PCR is able to distinguish reliably between the many Borrelia strains.  

WE PROBABLY ONLY DETECT THE "TOP OF AN ICEBERG" OF TRUE NUMBER OF BORRELIA INFECTIONS! - nobody knows how many of common chronic illnesses that might have an infectious origin - perhaps Borrelia, plus minus other microbes?  - and we will never find out more about the truth, as long as we do not have access to use all the Borrelia test methods that currently available, especially the DIRECT METHODS OF DETECTION OF BORRELIA, culture being named the GOLDSTANDARD diagnostic method.

8 years later (1990) Schutzer et al. in found circulating immune complexes in Lyme borreliosis explaining the patients false seronegative status, PMID: 1967770 from Abstract:
"These findings were confirmed by western blot, which also showed that immune complex dissociation liberated mainly antibody reactive to the 41 kD antigen [flagella] and sometimes antibody to an approximate 30 kD antigen [ospA] . Complexed B burgdorferi antibody was also found in 21 of 22 (95%) of seropositive patients with active disease, 3 additional seronegative but cell mediated immune reactive patients, and 3 other seronegative patients who eventually became seropositive. Apparent B burgdorferi seronegativity in serum immune complexes may thus be due to sequestration of antibody in immune complexes".

Same authors published on this subject again in 1999 PDF from abstract:
"The B burgdorferi immune complexes were found in 25 of 26 patients with early seronegative erythema migrans (EM) LD; 105 of 107 patients with seropositive EM LD; 6 of 10 patients who were seronegative with culture-positive EM; 0 of 12 patients who were treated and recovered from LD; and 13 of 13 patients with neurologic LD without EM. Among 147 controls, B burgdorferi immune complex was found in 0 of 50 healthy individuals; 0 of 40 patients with persistent fatigue; 0 of 7 individuals with frequent tick exposure; and 2 of 50 patients with other diseases...

- same has been confirmed by many other authors, a selected reference list in this article (in danish) by Marie Kroun.

Other - especially serology diagnostic - problems is raised by antigenic variation - explained by Barbour - by intracellular location, by the bacteria using host factors, for instance coating its surface, which have receptors for host factors ~ also acting as antigens (stimulation immune responses), and interacting with host proteins like fibronectin (PDF), plasmin/plasminogen (PDF, PDF, PDF), integrins (PDF), matrix metalloproteinaser (PDF), decorin (PDF, PDF, PDF), glycosaminoglycan (PDF) ... more?), thus enhancing penetration over endothelial and other membranes, protecting itself from binding and degradation by complement activation, and hiding it surface antigens, changing into alternative forms without cell-wall and thus without membran associated surface antigens (no flagellin exposed = no stimulation of anti-flagellin antibodies:  PDFPDFPDFPDFPDF, PDF, PDF), thus abrogating further immune anti-flagellin stimulation and also rendering already formed antibodies useless, because the surface antigens are either not the same anymore or are blocked / hidden behind host factors, by entering deeper tissues like tendon/ligaments, inner eye, discus with few or no blood vessels to transport immune-cells, antibodies and antibiotics to the place; Borrelia is microaerophilic and can survive in such places with low oxygen! - so there they can hide safely and later re-emerge, when the environment has become less hostile to the spirochete form (immune defences lowered) and not the least Borrelia's capability (in vitro) to enter and kill immune cells specifically those directed towards borrelia (PDF, PDF) ... a flagella less mutant of Borrelia is untouchable by flagellin-antibodies, making it able to survive and later re-develop flagella again when needed (PDF); while it is circulating in the blood stream Borrelia, blood cells, everything .. will be moved passively around by the cardiocascular circulation, thus Borrelia do not need flagella organs to move in the blood; since flagella is one of the first and highest immune reaction triggers, it must be an advantage for Borrelia to remove the flagella and other surface antigens, until they are again needed  .. non-motile spirochete like structures can sometimes be found in the blood by microscopy ... and other forms, see 2001-AdverseConditions.pdf
... everything on the microbial surface is there for a purpose, acting as receptors or anchors by which Borrelia may adhere to and penetrate membranes to bind host proteins that enhances penetration of host membranes etc. etc....  but may also act at immune stimulators and attack points (antigens).

Many many smart tricks have been developed, making Borrelia species, able to survive long term (for the rest of its mammal hosts life time?) under unfavorable conditions and they may probably persist for decennials in their mammal host, even despite fierce immune reactions are being raised towards Borrelia in the immunocompetent host!
Latest but not least bacteria communicate with each other and regulate synthesis of their proteins / growth by molecule-talking - Quorum Sensing - has now also been found for Borrelia, see Bonnie Bassler's video-presentation on how she discoverd Quorum Sensing here.
A few Borreliae that are only active on rare occasions, may not do much harm, but if many are present and active and "decide" to begin to form toxins - Bbtox1 for instance - then they may begin to cause many severe health problems ...

IT SHOULD BE PURE LOGIC TO ALL THAT ARE ABLE TO READ AND THINK, THAT NO SINGLE ANTIBODY TEST CAN FIND ALL BORRELIA, because Borrelia species is so smart and antigen variations occur all the time!
- we need access to all possible test methods! 

Borreliosis will remain to be a mainly clinical diagnosis, where weight is put on the patients known exposures to previous or recent tickbites, a previous or concurrent rash either at the tickbite-site or elsewhere, the waxing and waning course and often gradual spread of symptoms from in the beginning local symptoms (often located to joint or nerves near / on the same side as the site of tickbite or rash occrurred), over years gradual spread and development of multi-organ-manifestations, and the exclusions made of other more common medical explanations for the patients multiple symptoms; the clinical tentative Borreliosis diagnosis can be supported by any positive test-result pointing to Borrelia infection or previous exposure, but may not be outruled based on negative test results!   


DAKO compagny history
- is listed here because of the many name changes that must be kept in mind, when reading scientific publications using either DAKOPATTS - DAKO - DakoCytomation - DAKO or OXOID (microbiology) products: 

Founded in 1966 by a Danish medical doctor, Niels Harboe, Dako is headquartered in Denmark with manufacturing and research sites in Glostrup, Denmark and Carpinteria, California, USA.
Dako earned its reputation for innovation and quality by introducing the first conjugated antibodies for immunohistochemistry 40 years ago.
In the 1960s several companies were producing antisera, but the strength varied greatly, presenting significant challenges for hospitals, which had difficulties using the antisera for the evaluation of patient samples. Niels Harboe realized the importance of obtaining a supply of antibodies for analytical purposes that was of the same strength at all times. He founded his own company, DAKOPATTS.
A discovery in early 1967 made it possible to standardize the strength of antibodies in a usable product. Soon antibodies became a useful tool for hospitals, to the benefit of patients all over the world. Since then Dako has continously been a driver in developing the field of cancer diagnostics introducing pharmDx, Special Stains, Image Analysis and Pathology Workflow.

Corporate Milestones

2008 Dako’s Flow Business Unit in Colorado, USA has been sold to Beckman Coulter. Thereby Dako’s focus on anatomic pathology, tissue-based cancer diagnostics and reagents for flow cytometry is strengthened.

2007 Dako launches Dako Link - Integrated Workflow Solutions. Never before has Dako launched so many important products at one time. The Dako Link solution offers software, instruments, reagents and connectivity options customized to meet the needs of any pathology laboratory. Dako Link substantially improves cost efficiency and turnaround time in the lab by linking together the entire workflow.
Dako gets new owner. Private equity fund EQT V (“EQT”) has signed a definitive agreement to acquire 100% of Dako. The total consideration for the transaction is DKK 7.25 billion.

2006 Dako launches ACIS® III. Dako launches the newest addition to our growing portfolio of automated platforms, ACIS® III. With a new sophistication, ACIS® IIII is enhancing the lab’s workflow to include advanced image analysis benefits in a simple manner. Pathologists now have the capability to improve their workflow, turnaround time, and experience cost savings.
Dako's Microbiology business sold. The sale of the microbiology activities to Oxoid is part of Dako’s strengthened focus on anatomic pathology. Dako’s microbiology range will gradually be rebranded as Oxoid and products should be ordered via Oxoid.

2005 Change of name. The company changes its name from DakoCytomation A/S to Dako A/S.
Microbiology up for sale. DakoCytomation sharpens its focus on cancer diagnostics by putting its Microbiology activities up for sale.
Agreement on image analysis.
DakoCytomation enters an agreement with Clarient Inc. on the further development and sale of its ACIS instruments, which are used for image analysis. The agreement is part of Dako’s strategy to automate more areas of the workflow in the pathology laboratory and to offer more integrated systems to the market for cancer diagnostics.

2002 Acquires instrument business. DakoCytomation acquires the Artisan instrument business from CytoLogix Inc. Artisan is a state-of-the-art tissue staining instrument.
Merges with bioinstrumentation company Cytomation and becomes DakoCytomation. DAKO, the world-leading producer of cancer diagnostic reagents, merges with the Fort Collins-based bioinstrumentation company Cytomation Inc. The purpose of the merger is for the two companies to utilize their common competencies to further strengthen their position in the fast-growing market segments of the in vitro diagnostics industry. Cytomation was originally founded in 1988 in Colorado by one American and three Australian scientists who were convinced that the industry-standard flow cytometer could do a better job.

2001 Sells Boston Probes. DAKO sells Boston Probes Inc. to Applied Biosystems Inc.

1998 Launch of HercepTest™. The joint approval of Herceptin™ (Genentech) and HercepTest™ (Dako's pharmDx test) was the first example in history of a pharmaceutical product directly linked to a specific test. 

1992 Danish pharmaceutical company Novo Nordisk A/S invests in DAKO

1991 Acquires Novo Nordisk Diagnostics. DAKO acquires Novo Nordisk Diagnostics (activities in microbiology) in Cambridge, United Kingdom.

1990 DAKOPATTS changes name to DAKO

1980 Establishes subsidiaries. In the 1980s DAKOPATTS establishes more sales subsidiaries around the world.

1979 Enters the United States of America. DAKOPATTS Corporation is founded in the USA.

1966 DAKOPATTS is founded in Denmark. DAKOPATTS’ initial product series was purified polyclonal antibodies.