Video microscopy on blood from danish chronically ill patients
- with symptoms of chronic / relapsing Borrelia infection after antibiotic treatment

Microscopy done by Marie Kroun (phasecontrast, 1000X optical plus ?x digital) & Bela Bozsik (darkfield, 10.000X) on #1 and AP91

Video's are in WMV (can be shown with Windows Media Video player via Internet Explorer or VLC) format made with Windows Movie Maker; some computers may need allow activeX and/or to make * and * trusted websites in Internet Explorer security for the video's to show).
Note it may take some minutes to download part of the streaming videos, before they begin to play automatically, but once loaded you can rewind and play them again.
While the videos are loading, you can jump down to NOTE and read the text / explanation, later return to watch the videos play.
If you want to watch individual videos in full screen mode double-click on the video (ESC will return you to this webpage after viewing video in full screen mode) or right click on video when it is playing and use Zoom to full screen ...

Total time to watch ALL the videos < 40 min.

#45 2006/10/29 (106 Mb, 8 min., by MK)

#50 2007/04/11 (98 Mb, 8:30 min., by MK)

#23 2007/05/07 (33 MB, 3 min., by MK)

#1 2008/02/06 (55 Mb, 5:30 min., by MK) & 15+16-10-2008 (18 Mb, 2 min., by Bozsik)

AP91 2008/10/15 (11 Mb, 1 min., by Bozsik) & 02-12-2008 (4 Mb, 1 min., by MK)
(Note This pt. was not fat-fasting for 24 hours before the darkfield microscopy, some of the small dots are probably fat micelles reflecting the indirect light)

2008/12-2009/03 AP91 was treated with antibiotics and got well; the improvement lasted until 201009 clinical relapse, videomicroscopy 2010/10/29 (23 Mb, 2 min., by MK):

2012/10/18 Unfortunately - this patient has relapsed again (no new tickbites) highly symptomatic for 1 month; 20121018 (32 Mb, 2:05 min., by MK):

RM81 2010/08/06 (12 MB, 1:30 min., by MK)

SD68 2010/09/07 (45 MB, 4 min., by MK)

Microscopy of same sample buffy-coat blood smear stained with Diff-Quik and Acridin Orange:
This blood sample is taken AFTER previous Tx. with antibiotics IV (early 2010) and while pt. is currently on minocycline plus clarithromycin!

ON43 2011/11/09 (28 Mb, 3 min., by MK)

Written report in danish:
Preliminary history and notes (coming).

0018 2012/10/04 () - previous case history and previous test results reported at conf. in York 2004, see:
After Tx with doxycycline in 2001 and again after relapse in 2003 - which did not keep his recurrent weekly vasculitis attacks at bay - and because he was NEVER granted any treatment for ringforms (detected both in 2001 and 2003 by two different microscopists!) - the local university hospital denied to re-evaluate him after the relapse in 2003! - so in lack of atovaquone suspension, he was treated by MK with metronidazole and azithromycin, a combo that had helped some other patients with borrelia + ringforms in RBC gain better control; after that change he improved considerably and was able to stop antibiotic Tx in 2004; since then he periodially has been symptomatic with episodes of general malaise, however rather short episodes, not enough to warrant retesting and retreatment in the patients opinion, not until in 2012/09 he felt very bad worsening with more widespread vasculitis attack (though not as bad as in 2003 yet, no photo, will take if it recurs), plus general malaise, fatigue, headache, nausea and dizzy spells, aches and pains - all the usual ... His GP now has detected co-morbidity, besides his heavy overweight, has developed a slight hypertension and sign of type-2 DM; this is NOT SURPRISING, because the stress raise blood pressure (often only during attack!) and the proinflammatory cytokine TNF blocks insuline receptors ...

Long term and over many years repeated biotoxin exposure and cascade effects, create at lot of neurological, immunological and hormonal functional disturbances, that may become irreversible after long time (> 2-5 years it seems many reach a point-of-no-return to normal!) exposure to biotoxins, as investigated and described by Dr. Ritchie Shoemaker since circa 1997, see his mold website:
- especially the explanation of "The Biotoxin Pathway"; download the chart as PDF for better print quality:

Shoemaker R - biotoxin pathway (coming)

These above selected videos are presented in order to show DOCTORs with a special interest in diagnosing and treating chronic spirochetosis / chronic Borreliosis by doing microscopy themselves, what they need to look out for in the microscope.
MK only does microscopy as research on selected members of who - judged from their history, previous test results and not the least the patients current symptomatology, visualized on symptom scores and log curves via the Excel symptomdiary (english & danish version) -
displays a recurrent relapse pattern suggesting the patient could be suffering from a currently active Borrelia infection and/or other infections carried by ticks! - read more about the diary and curve drawing here:

Microscopy procedure / hints:

Bozsik's Dualdur reagent / dobbelt centrifugation method is described in #1 video from 15-10-2008 and more information here. Please also read more on the subject of 'why buffycoat'.
I (MK) take a few (3-5) 5 ml EDTA full blood samles, centrifuge one at high speed for 20 minutes, and isolate the buffy-coat fraction (store the removed plasma in the freezer for eventual later serology tests), and usually make 10-20 dried bloodsmears from the buffy-coat fraction (plus make a few additional unconcentrated bloodsmears for the differential count) and examine at least one wet drop (around 50 microliter) of the wet buffy-coat fraction meticulously,
always with the 100X oil objective, too see if there are any moving microbes therein. Meanwhile the smears are left to dry and can later be fixed, stained and examined for microbes.
The QBC-Paralens objective, can instead of its external UV-light source, also be used with the normal microscope light source
for doing ordinary normal light microscopy; then a quick switch between normal light microscopy and fluorescence microscopy is possible by just shifting the light source; that is handy when I am using an immunofluorescencent stain or acridine orange stain, it is possible to look at the same structure in different microscopy modes without having to move the object/viewfield; I have 2 sets of oculars, 6x and 10x, for the old Leitz Wetzlar Laborlux (III) anno 1957 microscope, thus I can "screen" a buffy-coat blood sample faster at a total of 600x magnification (as long as nothing abnormal is found), then if I encounter something that needs a closer look at, I can quickly increase the magnification to 1000x by shifting the 6x oculars out out with the 10x oculars and I can exchange the right ocular with the microscope USB camera, which I usually always do, because structures that are magnified further digitally on the computer screen are much easier visible, than they are by looking at them in the microscope, and besides this gives me a more comfortable working position, I can sit leaned back looking at the screen, while looking at the sample, which is much more comfortable than to sit bending my neck in order to look into the microscope oculars, where the very bright light directly in my eyes often are quite unpleasurable and provoke headache (I am often very light sensible); the camera also allows me to snap still pictures and do video sequences of any moving objects in the sample and often I have the camera running throughout the microscopy, in order not to miss a thing, but files must be broken into several max 1 gigabyte large video files, otherwise they can not be processed by Windows Movie Maker; also this way the patient and I can do the microscopy together, looking at exactly the same screen, when the patient wish to see what is found with their own eyes, which many do; my microscopic examination is always done very systematically in the same meticulous way. I always begin scanning the sample from one edge, scroll stepwise all the way up or down to the opposite edge of the sample, then step one viewfield sideways (in 600x), then scoll stepwise back to the opposite edge and so forth, and continue the procedure until the whole blood sample have been examined very thoroughly. It can take me several hours to do microscopy of a single sample, because I do not want to overlook anything. There may be very few microbes, sometimes only one, in a single drop of 50 microliter buffycoat blood sample; finding > 7 (~ many) abnormal structures per examined drop BUFFYCOAT fraction of a 5 ml EDTA blood sample is a rare finding in the patient group I investigate, that are chronically ill with borrelia plus eventual other tickborne co-infections.
If I do not find anything abnormal at all in the first sample screen, but the patient is highly suspect of having infection that flare up in cycles, I find it a waste of my time and effort to look more at that set of samples, because we probably did not hit the right day of sampling, i.e. during a microbial breakout; instead I rather will set the patient to do detailed symptomdiary in Excel (if not already started) and come for a new test another day, within 12 hours after start of a new flare in the borrelia cycle, and the day before a new anticipated flare up, for the intracellular blood infection that we want to look for just before the breakout of the microbes from the cells; finding very few microbes in circulation depends very much on if the sampling can be timed perfectly according to the patients symptom log; unspecific "infectious" symptoms break out within a few hours after microbes have broken out into the blood stream from deeper tissues (spirochetes) or from intracellular compartments (babesia, anaplasma, ehrlichia ..) and results in a fierce immune reaction with formation of lots of antigen-antibody immunecomplexes (in the immunocompetent host), so when there are many enough antigens entering the blood circulation at the same time, that bind antibodies to them, there may actually not be any free left over antibodies in the plasma/serum that can bind to the serology test antigen, i.e. the patient may very well test false seronegative in the very same plasma sample wherein antigens are detected by microscopy of the buffycoat by meticulous microscopy!
It is probably the formation of immune complexes that trigger innate immune system, both complement cascade reactions (measure complement split product C4a; C4a may be elevated in patients with Borrelia and long lasting rheumatological complaints PDF but was not in patients with borrelia dominated by neurological symptoms) and a proinflammatory cytokine cascade response (TNF, IL-1, IL-6 etc.), which should also be measured if possible, because
if there are abnormal innate immune reactions, it shows there is an inflammatory proces going on in the patient but not the cause, but if positive for inflammatory markers, we know for sure this patient is feeling sick for a reason that must be sought (it is not just in the patients head), and the patient may need antibiotic treatment to reduce the symptoms by suppressing microbial growth, hence abrogate the cause of the inflammatory reaction, if microbes can be detected by tests that are sensitive enough.
The patient may feel quite sick, despite there is not any
measurable specific antibody response, either because all antibodies get bound into immunecomplexes, the most of which probably precipitates inside the hosts vessels (hence vasculitis-like reactions, petecchia, easy bruising, declive edema indicating increased vascular permeability, are common complaint during periods of infectious activity), or because the patient is not able to mount a specific immune response for various reasons! - because it is cascade reaction, that can be started by very little amount of antigens - just like the hyperallergic patient can react very fiercely with an anaphylactic shock reaction to minute amount of antigens that does not bother a non-allergic person - the patient can be very sick, despite it is so difficult to find the microbial antigens in their blood!

Other investigators that are less meticulous than I in their sampling as perfectly as possible according to the patients cyclical relapse pattern and in their methods of staining (not using both conventional light and flourescence microscopy) and doing microscopic examination less meticulously, like looking only at 400-600x magnification on a thin and thick blood drop from capillary blood ,
taken from either finger or ear prick i.a. doing a conventional "malaria" screen, and who usually spend less than one hour in front of the microscope looking at the sample and perhaps only scan about 1/4 of a sample because they step 3-4 viewfields sideways instead of just one - may very easily overlook the single ringformed parasite or single moving spirochete that can actually be found in that particular patients samples - if they would do it like I do!
In 2000-2003 I compared the usual "malaria" screen technique with the buffycoat technique, and often found no sign of parasites in the conventional samples, but might find sign of increased bone marrow blood cell reproduction (normoblast i.e. immature red blood cells and leuco-blasts (immature white blood cells), as reaction to infection), in those patients, who turned out to show up a few parasites / microbes in their buffycoat bloodsmear; since then I only do examination of the buffy-coat for microbes, but might do an unconcentrated smear examination also, just to do an oldfashioned differential count.

I NEED A BETTER TRINOCULAR MICROSCOPE WITH DARKFIELD, PHASECONTRAST with SEPARATE VIDEOTUBE (c-mount), and a much better quality, very light sensible video camera (connect to computer via USB), that can take good pictures of even one single slim green spirochete on a very black background, and can magnify the picture from the microscope up to 100X!
- to get up to a total of 10.000X total magnification, so I can take videos of spirochetes in the good quality, as dr. Bela Bozsik and dr. Andy Wright can do
(see examples of their work on blood microscopy page from before 2006:
If you can spare some bugs, please support the research by donating to Patientforeningen DanInfekt (labelled research/forskning), see account number on at KONTINGENT subpage.

Please beware that finding spirochetes and their alternative structures by simple microscopy, as illustrated in videos above, is not a "test-result" that can stand alone
- since all spirochetes look pretty much alike in the microscope, and since all spirochetes may undergo the same (cyclical) developmental changes such as degranulation, formation of cysts, blebs etc., as illustrated in this 100 years pictorial "Survival in Adverse Conditions" and described by DeLamater 1951 and by Hindle 1912 and others, also read the historic observations on (relapsing fever) Borrelia spirochetes; so when the human eye and/or camera detect a few moving ("baby" or adult) spirochetes in a blood or spinal fluid samplefrom a human patient, it is not possible to say with absolute certainty which spirochete is in action, though a history of tickbite(s) aquired in the temperate climate zone followed by EM, point more likely to Borrelia burgdorferi sensu lato / Lyme borreliosis, than to another type of spirochete (Treponema (syphilis) or Leptospira (leptospirosis AKA Weil's syndrome, rat catchers symdrome or 7-day-fever and more ...)) .

Note that Willy Burgdorfer - the discoverer of
Borrelia burgdorferi - in his 1999 Keynote lecture (saved PDF printout from Medscape) was honest enough to admit that he was wrong way back in 1951 when he did not - as he writes / said - found evidence of "a negative phase or complex life cycle" ...

"Although most of the above mentioned opponents of the "granulation theory" verified the formation and existence of cysts, blebs, spherules associated with spirochetes, they considered them as degeneration products." ... "Of particular interest to our discussion is the presence in freshly engorged Lyme disease ticks of spirochetes with outer membrane-associated cysts, blebs or spherules that often contain numerous granules with surrounding trilaminar membranes (Figs. 11,12). Because the internal material of these granules is similar in appearance and electron density to that of typical spirochetes and because these cysts (blebs, spherules) strongly react when treated with FITC-labeled conjugates, the questions concerning a complex life cycle of borreliae have again been raised and induced investigators to critically examine the nature and function of these formations -- a research problem referred to for the first time almost 100 years ago by Dutton and Todd. Thus, RML scientists Dave Dorward and Claude Garon using silver staining, transmission and scanning electron microscopy investigated the nature of naturally elaborated membrane blebs on the surface of cultured B burgdorferi or free in the medium, and found both linear and circular DNA (Fig. 13). The fact that his material was packaged within the membrane-derived vesicles suggested that it might play a role in the protection of genetic markers. In vivo and in vitro exposure of B burgdorferi to antibiotics (penicillin G, ceftriaxon) were shown by Preac-Mursic and associates to produce cytomorphic atypical but motile spirochetes with numerous membrane-derived vesicles (spheroblast -- L-forms) (Fig. 14).[1]. In their recent publication, Brorson and Brorson reported on the "In vitro Conversion of Borrellia burgdorferi to Cystic Forms in Spinal Fluid, and on the Transformation to Mobile Spirochetes by Incubation in BSK-H Medium."[2] Accordingly, B burgdorferi converted rapidly to cystic forms when transferred to spinal fluid. No normal spirochetes were left after 24 hours of incubation at 37° C; all were converted to cysts. When these cystic forms were transferred to a rich (BSK-H) medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. [young cysts]" ... "Using silver impregnations and immunochemical staining, cystic material has been demonstrated in every animal and human tissue infected by B burgdorferi. As yet, it is not known whether these forms of Borrelia represent products of degenerated spirochetes or of surviving organisms capable of transforming to typical spirochetes once the faborable environmental conditions are restored. It is tempting to speculate, however, that the survival mechanism of spirochetes is responsible for the diverse pathology of these organisms as well as for their ability to survive as cystic forms thereby producing prolonged, chronic and periodically recurrent disease." ...

Hampp 1948 did electron microscopy studies on spirochetes and wrote:

"Typical free granules, the end products of granule 'shedding', are shown in figure 18. They are roughly circular in outline and sharply bounded. They consist for the most part of what appear to be short sections of spirochetes closely packed together. The contents of these granules are probably responsible for the fine lacelike appearance and the bright white, highly refractile bodies described by Hampp (1946) under the dark-field microscope. Examples of another type of free granule repeatedly observed are shown in figures 19 and 20. These granules consist of tangled masses of spirochetes or spirochetal segments. The significance of granules in the life history of the spirochetes is unknown but certain investigators have suggested that they may be germinative units (Balfour, 1911; Noguchi, 1911; Noguchi, 1917; Leishman, 1918; Mudd et al., 1943; Hampp, 1946). Others are undecided or hesitant in accepting this hypothesis (Fantham, 1916; Akatsu, 1917; Wenyon, 1926; Warthin and Olsen, 1930). Topley and Wilson (1936) have indicated that they are probably particles of culture medium adhering to the sides of the spirochetes. The electron micrographs demonstrate that this explanation is wrong, and that free granules are definitely a phase in the development of spirochetes. Although it is not possible to determine from these micrographs that the granules are germinative units their constant rhythmic occurrence in living cultures suggests this possibility. Further support of this hypothesis is provided by the fact that cultures up to 31 months old, showing only refractile granules by dark-field examination have invariably given normal growths on transfer to fresh medium (Hampp, 1946) ...

... 31 months ~ 2½ years! - muchlonger than the duration of the usually for spirochetal infection recommended antibiotic treatments!

The latter question mentioned by Burgdorfer, if cysts can convert back to spirochetes also in vivo, have later been answered by Gruntar et al. - APMIS 2001. PMID: 11478686 - who injected variably aged cystic form of
Borrelia garinii intraperitoneally in mice, and found it could result in Borrelia infection, judged both by seroconversion of the mice and by finding spirochetes in the heart and bladder tissues of the with Borrelia cysts experimentally inoculated mice, when they were sacrified and dissected at end of the study!

In order to be able to put a family name / substrain name to the spirochete seen in the microscope, we'll need to do specific and unfortunately expensive supplementary ANTIGEN TESTING, depending on which spirochete we suspect it can be, judged from patients history and previous test results, and depending on what is availble on the marked to buy for supplementary testing, at present it is possible to buy FITC-labelled affinity purified Borrelia burgdorferi antibodies from KPL and ready to use solution of BSK-H medium from Sigma-Aldrich.
While it can be quite difficult and time consuming to get positive result with specific direct immune stain or PCR test when there are only a few spirochetes present in the material (usually less than 5 spirochetes are found per buffy-coat drop) i.e. lower than the detection limit for PCR? - it should be possible to increase chance of these methods giving a positiv result,
if we can first get a positive culture of spirochetes, so there will be much more spirochete material to do PCR or specific immune stain on, thus less waste of time and expensive materials on "nothing found" and the chance of getting a positive culture should increase, when one can first find spirochetes by a simple microscopy screening like above, that cost some hours of work but not much money.
So far I've already got some positive cultures, already in the first few tries; preliminary investigations for a future DanInfekt Borrelia culture project, where we will attempt to culture in BSK-H as explained by Barbour and others in my selected:; it is probably quite important to avoid oxygen mixing since Borrelia are microaerophilic and to keep the optimal temperature around 33-36 degree Celciis, for increasing chance of growth of Borrelia: ... in case of a positive culture, we can split a serum sample taken from same prick / at same time as microscopy as the blood for culture and do WB (like Mikrogen Borrelia recomBlot or Euroimmune Line blot) abroad and
DAKO/OXOID Borrelia flagella IgM and IgG antibody analysis on the plasma/serum, for a direct comparison, a similar study to what Oksi et al. did in JCM 1995, se ref. below. If we can succeed in culturing individual patients own spirochetes in abundance in vitro, such cultured spirochetes can then be used as antigens in a patient specific indirect fluorescence assay, to test if the patient has formed any specific (IgG) antibodies that react with the spirochetes grown from the patient. It just requires either peroxidase- or FITC labelled anti-human-IgG antibodies, that can be added to make eventual antibody-antigen reaction visible. Simple.

Please note: immunoflourescence can be used both ways to detect either antigens or antibodies.
In the primary or
direct immunofluorescence assay (d-IFA or DFA) uses a single (microbespecific) antibody (achieved by vaccination of a clean laboratory animal which serum is harvested, purified by gel electrophoresis and absorbed for irrelevant cross-reacting antibodies to other microbes we do not want to see reaction with) that is chemically linked to a fluorophore like fluorescein. The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy.
In the
indirect immunofluorescence assay (i-IFA, or many just write IFA, the missing i- or d- in front may unfortunately confuse a lot of people) two antibodies are in the reactions; the unlabeled first (primary) antibody (within the patients serum/plasma sample) specifically binds to the target molecule on the test antigens; then a secondary antibody (anti-human-IgG antibody), which carries the fluorophore, recognises the primary antibody and binds to it, thus visualizing that some IgG within the patients serum/plasma had bound to the test antigens residing on the plate. Instead of fluorophore the secondary antibody can carry an enzyme (for instance peroxidase) that when reacting with a suitable substrate create a visible / measurable color reaction, as in the ELISA AKA EIA.
Try doing the ELISA and PCR procedures yourself playing a laboratory worker in this virtual laboratory (interactive video lectures).

Many doctors continue to claim there is not sufficent proof of persistent infection in "chronic Borreliosis / chronic Lyme disease" - this can only be because the people who claim that has not read the literature on by direct detection method proven persistent Borrelia infection - to help find the articles and cases I long ago (first put on Internet 2003) made a reference list
Persistent borreliosis / relapsing Borreliosis verified by direct detection methods culture, microscopy, PCR:

MK noticed that some Bowen test project participants developed neuroborreliosis / later relapsed and suffered from chronic/relapsing Lyme borreliosis (verified by microscopy with DFA i.e. Direct Fluorescent Antibody stain for Borrelia burgdorferi)
- despite they had had what is normally considered to be "appropiate" early antibiotic therapy for erythema migrans

- and moreover confirmed relapses / progression of disease were seen, both after penicillin and after doxycycline in officially recommended doses!

Below focus is set on some publications on European culture or PCR verified LATE (> 3 month duration) cases / or relapses
- and
DAKO (OXOID) serology result!
- which is of special interest to MK / danish patients, because the DAKO test was developed in Statens Serum Institut (SSI), Copenhagen, Denmark - by Hansen, Lebech and DAKO - and is still the ONLY test for Borrelia in use in Denmark, however since 2006 under name OXOID

- read more about it in:

Unfortunately danish microbiologist still refuse to to microscopy (enhanced with acridin orange or specific direct immune stain for Borrelia), culture nor will they do PCR for Borrelia, nor a WB - despite new recombinant multi-antigenic, rather cheap WBs are now available, where only the diagnostic relevant bands are selected, i.e. the result is easy to read, creates less confusion, since most selected bands on the test strip are Borrelia specific!
They state they find these methods too laborious, too time consuming, too expensive for the lab to run them - however these labs do NOT take into consideration, what it costs the patient not to get diagnosed and remain ill for years / lifetime from Borrelia infection, not take into consideration what it can cost the danish health system - since these patients go to many specialist, who must each evaluate the chronic multi-organ-system symptomatic patients for a lot of diseases that all come out negative/normal, because that is NOT diseases that this patient have, nor was it very likely, judged by the history pointing to Borrelia infection
- but doctors rather tend to do "defensive medicine", i.e. do all the ROUTINE tests they traditionally do on most of their patient to "outrule disease within our field of medical speciality", and when they have done that, the doctors feel they've done alle what they can / must do "within their speciality", despite they have not helped the patient to get well - none of them can't (or won't do) the test which the tickbitten patient really need most of all, multiple tests for tickborne infection, for immune dysfunction etc.!
- and they do also not consider either how much it cost the danish social system, when the long-term ill patients are way too sick to work, but can't get a diagnosis / prognosis and must go on transfer income paid over our taxes ...

The laboratories that is paid by out tax money should SERVE the DOCTORS and their patients with the BEST available tests on the marked, not what they self developed long time ago, nor what they find is the easiest for them to do!
WE PATIENTS FIND IT A BIG WASTE OF OUR TAX MONEY THAT THEY ONLY WILL DO A PROBLEMATIC ELISA SEROLOGY TEST FOR BORRELIA THAT WAS SHOWN (Oksi 1995) TO MISS NO LESS THAN 58,5% OF CULTURE OR PCR PROVEN LATE CASES OF BORRELIA INFECTION, THAT IS BASED ON A SINGLE NON-SPECIFIC ANTIGEN, FLAGELLIN, THAT 20 YEARS AGO (Picken 1992) WAS SHOWN TO VARY GENETICALLY ENOUGH TO BE ABLE DIFFERENTIATE BETWEEN THREE EUROPEAN BORRELIA SUBSTRAINS ... gene variability => antigen structure variability => antibody structure variability! => a test based on flagella from ONE strain of Borrelia, is probably less able to bind antibodies formed against different strains of Borrelia? - pure logic could and should have been applied byt the test producers already in 1992 and no later than 1995! - but instead of recognizing the problems with their test, they chose to ignore literature speaking against their serology test!
Regarding Borrelia serology test I prefer using
Line immunoblot (either Mikrogen og Euroimmun), because they are as highly specific as ELISAs on the marked, but more SENSITIVE than any Borrelia ELISA on the marked! - and is easy to do in the laboratory (run automatically after reagent have been added to apparatus, is measured by computer - little risk of human errors!

J Clin Microbiol 1995 Sep; 33(9): 2260-4.

... 3 serology tests evaluated on 41 late (> 3 mdr.) culture or PCR verified Borrelia cases
- amongst them DAKO / OXOID flagella ELISA came out false serum antibody negative in 24/41 (58%) cases with symptoms of Borreliosis for at least 3 months duration!
SSI failed to repeat a similar study in Denmark on danish patients with late culture confirmed Borreliosis and moreover IGNORE DISCUSSING / REFERENCING THIS IMPORTANT STUDY IN THEIR "guidelines"
- in DanInfekt we intend to try to examine OXOID serology in culture verified chronic Borreliosis cases, and compare OXOID result with WB ....

Strle et al. Clin Infect Dis. 2006 Sep 15;43(6):704-10.

... in spinal fluid culture verified Borreliosis, 23 w/Borrelia garinii, 10 w/Borrelia afzelii
- in the Borrelia afzelii neuroborreliosis group 9/10 tested negative on DAKO/OXOID neuroborreliosis kit, hereof had only two been sick for shorter than 6 months
- i.e. 7/10 (70%) had been sick for at least 6 months and up to seven years, with wrong or no (?) diagnosis, and those CSF ELISA Borrelia index. neg. would not have gotten the correct borreliosis diagnosis, if serology had been the only test for Borrelia done (like would be the case in DK, where microbiologist, SSI will NOT do culture nor PCR test for Borrelia on chronic (seroneg.) patients!

Honegr et al. Cent Eur J Public Health. 2004 Mar;12(1):6-11.

... IgG and IgM titres to the Lyme spirochete were determined by ELISA using standard conditions as recommended by the supplier (Test-Line Ltd, CR).
In ELISA with sonicated antigens, a positive value was defined as 0.73 for IgG and 0.850 for IgM.
In the IgM capture ELISA (The 98th percentile of absorbance values for the controls was used as the cut-off level. Dako Diagnostika GmbH), a positive value was 0.380.

Compared IgG serology measures and which treatment failed first (from table 3 and 1), for IgM look at full text:


First treatment before
1st detection of Borrelia antigen
(from table 1)

1st/2nd detection



neg. / neg.



ROX / 4

neg. / neg.



DOX/ 7

neg. /neg.



pos. /pos.



DOX / 10

neg. /neg.



DOX / 12

neg. /pos.



pos. /neg.



pos. /neg.



neg. /neg.



neg. /neg.



DOX / 16

neg. /neg.



neg. /neg.



DOX / 9

pos. /pos.



pos. /pos.



neg. /neg.



neg. /neg.



DOX / 12

neg. /neg.



neg. /neg.


Regarding CUT-OFF values confusion - just for comparison:
Dako Glostrup cut-off values, also used 98 percentile as cut-off level, but cut-off level has changed over time, and apparently without scientific justification, since it seems no new validation study is searchable PubMed in between the following article publication dates nor at a later date?!
Ann Neurol. 1991 Aug;30(2):197-205. Cut-off for IgM: 0,300 vs. IgG: 0,160 (sent in for publ juli 25. 1990).
J Clin Microbiol. 1992 Jul;30(7):1646-53 (PDF). Cut-off for IgM: 0,500 vs. IgG: 0,160 (received Jan 1992) [IgM cut-off increased with factor 1,6]
Neurology. 1993 Jan;43(1):169-75. Cut-off for IgM: 0,500 vs. IgG: 0,240 (received Apr. 6, 1992) [IgG cut-off increased with factor 1,5]

=> Why / what justified the increase in cut-off values by in 1992?

Increased cut-off value means the antibody level need to be ~1,5 times higher in order for the patient get a positive Borrelia antibody test result!


Note that Honegr et al. - when looking at IgG - found a discrepant / unexpected discrepant second negative ELISA IgG results, when compared to WB!
- a pos. WB but neg. 2nd ELISA IgG is marked with bold in table:
12/18 (2/3, 66%) were IgG ELISA neg. but were WB-IgG-positive, and since they all had positive detection of Borreliae by either PCR or IEM, it is the WB result that was the TRUE POSITIVE RESULT! - and the neg. IgG ELISA was false negative result!

NOTE that pt. #7 and #8 both developed negative IgG ELISA result, despite they had positive IgG at first test, and despite positive WB!
- like other cases on the persistent borreliosis reference list with by direct method detected persistent borrelia infection post treatment, at time of clinical and confirmed relapse!

NOTE that 6 /18 (1/3, 33%) relapsed despite they were treated before the first detection of Borrelia by IEM or PCR with doxycycline
...and one had roxithromycin, a drug earlier known to fail (1992, PMID: 1357894) - maybe this pt. was treated with roxithromycin before publication since pts. were enrolled / detected between 1991-2000?

- so lets look at some probable reasons, why so many patients relapse after doxycycline ...

Borrelia has been detected by PCR very early - within the first 2 weeks after the tickbite - in the central nervous system (CNS), i.e. before and without later development of an erythema migrans:
Therefore it does not really make good common sense to treat erythema migrans with drugs like peroral penicillin, that can not penetrate over membranes into the CNS
- nor does it make sense to give a low dose / shorter duration treatment for an EM, than what is recommended for treatment of neuroborreliosis!

Thus the Bulls-Eye rash, Erythema (chronicum) migrans (EM / ECM), is a side-manifestation, that may sometimes occur, but it is not a prerequisite, that EM must occur before Borrelia can spread into the CNS and cause neuroborreliosis! - around half of patients later diagnosed with neuroborreliosis never saw an EM rash!

Problem is that treatment with a sub-inhibitory / sub-lethal dose / drug concentration will not kill all Borrelia, but will select the most resistant of the bugs, which later are those that cause the relapse, and will need either another drug or a higher dose for the bugs to be sensitive and be killed!
Therefore a second antibiotic treatment with same drug and dose often does not show as much clinical effect as the first treatment, unless using other drugs with very good penetration to CNS, and/or if a much higher dose is used!

Lack of a good response to retreatment is often evaluated as if the chronic / relapsing symptoms can not be caused by an ongoing Borrelia infection; however, when searching for the microbes by direct method like microscopy we can actually find the spirochetes again and again - as illustrated in videos from different patients above - especially in patients that has turned seronegative after the first treatment, since many or all the antibodies they form, during a relapse with so many bacteria in the blood stream that they can be detected by simple microscopy, are quickly used up by binding in to all the circulating Borrelia antigens, which precipitate in vessels, cause vascular damage and vasculitis with chronic inflammatory cells, the micropathological Hallmark of Borreliosis (and syphilis)! - elicit complement cascade, proinflammatory cytokine response, like increased TNF-alfa ... it is the immune reaction that cause the symptoms ...
Serology can detect early infection only several weeks after aquiring the infection, when serial serology tests confirm a positive seroconversion, and in late infection can detect past exposure to Borrelia antigens or alike when positive
- but since IgG serology can stay high for years in patients with previous Lyme borreliosis (who do not have circulating antigen to bind the antibodies) who became asymptomatic on given antibiotic treatment, serology really has no use whatsoever in the diagnosis of chronically ill patients of longer than 3-6 months disease duration!

A negative serology test can not not outrule Borrelia infection; pt. may be immune depressed or have so much circulating antigen that all antibodies are bound in the patient so nothing is measured by the test!
A positive serology can not prove the patient have a currently active infection!
- and such patients should be investigatede thoroughly, not be dismissed as if they are malingering hypochondriacs!


Treatment with (low dose) doxycyclin for Borrelia infection fails very often (like in #45, #23, #1, #50 and RM81 above!)
... the only patient above who did not relapse after doxycycline was AP91, who was a child and therefore got penicillin, not doxycycline, for her first EMs! - still she relapsed despite penicillin in the usually recommended EM dose, not only for the 10 days that is usually recommended here in Denmark, but for 6 weeks, as suggested by Burrascano!
- see the latest version of his guidelines at

More of 50 project participants relapsed despite early recommended treatment for their EM !!!
probably because the usually recommended dosis of doxycycline 100 mg x 2 does NOT REACH A CONCENTRATION OVER THE MINIMUM INHIBITORY CONCENTRATION (MIC) IN CNS and other "remote areas"?!
- since doxycycline distribute well in fatty tissues, especially fat people 100-150 kg will have 2-3 times larger distribution volume of the drug, hence will reach a lower tissue concentration on the same dose that give sufficient concentration in the small 50 (-70) kg person: (some text is in danish, but references and abstract is in english, use Google translate!)

The actually achieved steady state concentration of doxycyclin was measured in spinal fluid by Dotevall & Hagberg 1989 (PDF)
- and they even used a lower MIC of only 0.6-0.7 micrograms/ml, where others state that MIC for doxycycline for Borrelia is 1 or even 2 micrograms/ml!

A dose of 100 mg of doxycycline (Vibramycin; Pfizer Inc.) was taken orally b.i.d. for 10 days by 10 patients, while 200 mg was taken orally b.i.d. for 10 days by 12 patients. On days 5 through 8 of treatment, CSF was collected 2 to 3 h after the last drug administration. In one patient, an intraspinal catheter was inserted for treatment of severe chronic pains, and repeated CSF samples were taken through the catheter during the doxycycline treatment. On the day of lumbar puncture, the patients were questioned regarding adverse reactions to the drug. No other antibiotics were given simultaneously. ...
Dotevall measured doxycycline conc. in CSF
Please note that NONE of the patients on doxycycline 100 mg x 2 and even some of the patients on 200 mg x 2, did NOT reach a concentration in spinal fluid (CSF) over 1 microgram/ml
- which many state is the MINIMUM INHIBITORY CONCENTRATION for doxycycline for Borrelia!

Honegr. does not tell us dosis and duration of the given treatments, however, if the patients - all got the usually recommended DOXYCYCLINE dosis of 100 mg x 2 daily

WHO WAS RESPONSIBLE FOR THE CHOICE? - AND WHY WAS A DOXYCYCLIN DOSIS OF DOXYCYCLINE 100 mg x 2 chosen to be the recommended dose for treatment of EM and for (early) neuroborreliosis
- when Dotevall & Hagberg already in 1989 had showed by their experiment that 100 mg x 2 may not result in a concentration above 1 mg/ml in ANY patient!?

- who are NOWADAYS TO BE MADE RESPONSIBLE for continuing to maintain the obviously bad advice of recommending too low doxycyclin dosis for EM and neuroborreliosis?

THERE IS OFTEN NOT (as often stated in the media) ANY SAFE 24 HOUR PERIOD / A DELAYED TRANSMISSION OF BORRELIA, and TBE can be transmitted within ONE HOUR after tickbite started: ...

Strle Clin Infect Dis 1996 Jul; 23(1): 61-5. PMID: 8816130

25 of 35 (71%) of patients who remembered the duration of the infecting tickbite got a culture confirmed erythema migrans, despite less than 24 hour tick attachment time!
9 of 35 (26%) even got infected with Borrelia via a tickbite of less than 6 hours duration!

Around 1/3 of Ixodes ricinus ticks have been found to have a systemic Borrelia infection and may thus have Borrelia in their saliva glands all the time, and can thus transmit Borrelia immediately when they begin spitting; systemically infected female adult ticks may also have Borrelia in their eggs, thus can transmit Borrelia to their larvae transovarially / vertically, i.e.
tick larvae can be infective despite they have not yet had a blood meal!

Experts who continue to inform people incorrectly / out-of -date, who does not give a balanced review of all published literature so far on subject tickborne infection to the public
- should be held economically personally responsible for their actions
- by those patients who do not remove the tick within 24 hours due to misinformation, get infected and thereafter must suffer the consequences, by developing debilitating disease
- because they got bad/wrong information from the official sources, or who was denied testing and treatment by ignorant doctors!


by informing people that SOME TICKS CAN TRANSMIT BORRELIA IMMEDIATELY WHEN THEY BEGIN SPITTING IN THE WOUND DIRECTLY INTO THE CAPILLARY THE TICK SUCK BLOOD FROM - i.e. tickborne infections are probably spread hematogenously right from start of the tickbite!
- see the nice tick bite pictures and animation at LDA-UK website: ...

- after photographing the tick in situ with date on the picture as documentation for the bite!
- after marking the bite site, so you can look out for development of a growing red rash / erythema migrans.
Only this way can all tickborne infections and disease from these agents be totally prevented, the person will not need tests, nor treatment, will not have sick days etc.!

- since the split products from complement activation may increase within 96 hours after a tickbite in patients that developed signs of Borrelia infection / seroconversion ...
Borrelia serology will NOT become positive so early in Borrelia infection, unless the patient got reinfected with Borrelia and had memory immune cells that can raise a very quick serology response!

The Complement system is the first line and innate unspecific immune defense reaction raised in infection, but note increased split products does not tell for sure which infection / inflammatory proces activated the complement cascade! HOWEVER IT MAKES GOOD SENSE TO ME TO TREAT PATIENTS WITH RECENT TICKBITE AND INCREASED COMPLEMENT SPLIT PRODUCTS WITH ANTIBIOTICS, BECAUSE EARLY BORRELIA INFECTION CAN BE CURED BY SUFFICIENT ANTIBIOTIC TREATMENT!
- while chronic borrelia infection > 3 months and of many years duration is very hard to cure, meaning the patient can improve very much while staying on antibiotic treatment and may even become asymptomatic, but relapses are common after stopping the treatment! - because dormant cysts can not be hit, since the microbial metabolic processes with which antibiotics interfere are not active during dormancy!

- then eventual few Borrelia that might have reached CNS / CSF already may be killed also and those killed effectively, will not grow into many under a sublethal antibiotic concentration, that select the most resistant bacteria!
Undertreatment is worse than no antibiotic treatment, because treatment removes antigens from the blood stream (less immune stimulation) without clearing all the most "remote areas" for bacteria, i.e. undertreatment will both switch off the maturation of the immune response and will select the most antibiotic resistant microbes from the surviving population especially in "remote areas", from which new growth may arise under favourable growth conditions, if the patients immune system is not able to suppress growth, without help from antibiotics!

DURATION OF TREATMENT MUST BE OPENENDED, the goal is to cure the patient!
Be aware that Borrelia grows very slowly and may have very long dormant periods where they can not be hit by antibiotics, until they enter growth phase again, perhaps at long intervals (often as monthly relapses) ...

... it is both fast and cheap, if you do it yourself ... you have a probable diagnosis when you see the first spirochete swimming!
- and if the blood looks perfectly normal in a microscopy done during a symptomatic period, you should rather seek for another diagnostic explanation for the patients symptoms, than an active Borrelia infection! - and in that case you do not need to waste more energy, time and money on doing the more difficult / more expensive test methods for Borrelia!

Note in the old days detecting spirochetes by microscopy in a syphilic person combined with the clinical picture was enough for a tentative diagnosis of syphilis and was enough justification for the doctor to treat the patient with antibiotics! - why is that not so today also for Borrelia, when we can detect spirochetes in the blood?

- at least always re-examine the blood before intention to pause antibiotic treatment!
Don't pause the treatment if the blood look abnormal during ACTIVE PHASE (day one of a new relapse)!
If no relapses are visible for 6 weeks in the diary and the blood also looks very clean, pause antibiotic treatment under surveillance!
- i.e. THE PATIENT SHOULD CONTINUE THE SYMPTOM DIARY for at least 3 months after stopping treatment; because an early relapse will show as re-beginning flare cyclicity (usually weekly) and the symptom score will gradually increase along as more and more spirochetes are born, make toxins, are being killed, produce waste products, elicit cytokines etc. ...
- then re-examine the blood and re-treat if finding spirochetes again and related structures!

For advice on treatment read Burrascano's guidelines at ILADS, he's got experience since mid 1980ies from more than 10000 Borrelia patients, and he published guidelines for 20 years, and he know this infection better than most ... because he's had borrelia infection himself, like myself!

Finally, getting Borrelia infected yourself and having to find out how to diagnose and treat the tickborne infection(s), because you get rejected by illiterate and ignorant doctors
- is the very best teacher!

YOU are very wellcome to get some of my blood infused (#1), if you - after seeing the videomicroscopies and read this - continue to maintain your stance that Borrelia is very easy to cure, since - if you are right - you have nothing to fear by doing this experiment!
Doctors did successful transfer experiments, including on themselves,
of the skin changes since known to be caused by Lyme Borrelia species - erythema migrans (EM), lymphadenomatosis benigna cutis (LABC), acrodermatitis chronic atrophicans (ACA) - after penicillin treatment had proven very helpful since 1946; for selected historic "milestone" references, see my Borrelia history lecture: Hull 2001; Kassel 2006.

... read, learn, look in the microscope, revise your stance and treatment policy!

- this document beyond any reasonable doubt that we need better DIRECT DETECTION METHODS (culture like Advanced Labs in USA now offer) because serology test fails in 50+% of these cases, are week and fluctuating, often only weak positive after 2-3 months treatment has gunned the spirochete form down, so no antigen enters serum / CSF and bind nearly all the antibodies in immune complexes and thus remove them from measure with antibody test (no matter which antibody test, but depending on where cut-off is set), plus immune inhibititory factors ...

- because we have learned so much about what do not work and some of what works, but there is probably much more we need to learn :)

Research done by Eva Sapi's Lyme research group at New Haven (in co-work with Dr. Alan MacDonald) has shown for certain, documented by microscopy pictures, how Borrelia burgdorferi, besides the spirochete form and long known alternative cystic and granular / round forms (100 years pictorial PDF of spirochetes and alternative forms!), which can actually be seen both in light and dark field microscopy (see 1951-DeLamater below), also can form BIOFILM communities under adverse conditions for the spirochete form, plus antigen variation, plus coating surface with host factors etc. etc. - all the smart tricks the spirochetes can do!
In a very elegant culture - live microscopy study, using
BacLight fluorescent viability stains marking alive, cell wall intact, bacteria green and dead, cell wall defect, bacteria red, they investigated the effects on the 3 different forms of Borrelia, of two herbal antimicrobials Samento (TOA free cats claw) and Banderol - that had shown very promising effect in Horowitz test of the Lee Cowden protocol (presented at ILADS 2007 and later, ILADS-EU conf. in Augsburg 2011) .. compared with Doxycycline.
The work was published in Townsendletter (July 2010):
They found that
both Samento and Banderol, and especially when both were used together, could hit not only the free spirochete form, the cystic / round forms, but also the forms hiding behind biofilm, while DOXYCYCLINE only had effect on the spirochete form, and forced Borrelia into producing many more cystic / round body forms, than did both Samento and Banderol!

These findings can readily explain, why so many patients relapse after conventional antimicrobial treatment courses - even after long term treatment - with for instance doxycycline alone - all examples herein except for AP91, who never got doxycycline, because of the observed failure rate - she had rather long fairly good periods after metronidazole plus azithromycin for 6-12 weeks, BUT SHE RELAPSE in autumn every other year!
We already know from previous studies by several other researcher that other antimicrobials also only has effect on the spirochete form, but produce more cyst / round forms, that later come back with a vengeance, after stopping the antimicrobial treatment! - we are awaiting further treatment study with the Cowden protocol!
Richard Horowitz, MD in New York State has found the Cowden Support Program to be effective in markedly improving the condition of 70-80% of the advanced Lyme Borreliosis patients with co-infections over 4 to 6 months time, even if the patients had previously failed to improve on multiple courses of antibiotics. Dr. Horowitz presented the findings of his use of the Cowden Support Program in a fairly large group of his patients with Lyme Borreliosis & co-infections at the ILADS conference in the fall of 2007.
One danish patient I know and have followed for over 10 years now, was very near to death just a few years ago, but has returned to a much better life now on treatment by Lee Cowden :)
The best is that the herbal remedies are relatively non-toxic and well tolerated for long term treatment (6 months); hundreds of years of experience from traditional treatment in South America Indians show it is safer than the safest antibiotics, BUT THEY CREATE A HERX, and dose must be started low and increased gradually, as all other drugs that works for Borrelia!

Effects of all other antimicrobial drugs for Borrelia as well as for other microbes, should be studied in the same way!

Perhaps combine with (some of) Klinghardts advice, depending on which additional problems the chronic patient has ...

The Klinghardt Protocol (based on over 900 successful treatment cases)

The treatment of Lyme disease requires 4 distinctive steps:

Decreasing toxic body burden/unloading the system

Klinghardt Biological Treatment of Lyme Disease
Herbs Used in Treating Lyme Disease
Klinghardt Neurotoxin Elimination Protocol

The patient group I follow in DanInfekt is way too small, and not homogenous enough, to be able to conduct true science studies on treatment comparison!
- can only be a few more or less succesful case reports - so I do not study treatment options, but DIRECT DETECTION METHODS.
I have been waiting to see studies, like the one done by dr. Eva Sapi's group, that convincingly document the effect of the natural remedies Samento and Banderol on spirochetes and alternative forms and biofilm - when tested in the laboratory.
I have awaited to hear results of larger ongoing clinical treatment trials, conducted by Dr. Horowitz and others. FIGURES, CALCULATIONS, PEER REVIEW PUBLISHED.
I have awaited to hear personal good stories from people I have known personally long before; it was just few months ago, that I got the first very promising report from a danish case on the Cowden protocol
- a movie is underways, enjoy ...

All treatment should be started as early as possible after start of a relapse, to see if the duration of the relapse can be abrogated compared to what the patients previous experience with duration of relapse is, of course documented by symptomdiary; if treatment is not started until severel months into a relapse, near to usual spontaneous improvement, the positive effect observed may have not resulted from the treatment, but was the natural course, a spontaneus remission occurred ... THEREFORE patient need do the diary and observe themselves long enough to know their patterns and can tell the difference under treatment ...
The relapsing remitting course of borrelia and their very slow reproduction, make it difficult to evaluate treatment in an optimally constructed science study.
... if we can catch and culture the spirochetes reliably by improving our techniques as illustrated herein (which is not new, but copycat many other researchers descriptions! - the findings are consistent!)
- then we can use Sapi's method to test the actual microbe that the individual patient harbor for antimicrobial susceptibility - and can tailor the treatment better to what has a chance to work in that particular patient! - just like it is routine in most other microbe infections ..

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