What is wrong with microscopy in Borreliosis?

Nothing, as you will see from the below evidence.

Microscopy in diagnostics

Microscopy played a great role in identifying unknown pathogens and it is still the first line of diagnostics in some diseases where the novel diagnostic tools failed first time or did not prove to be efficient in the long term. Examples are syphilis and tuberculosis, less specifically, the scanning for urinary tract infections is done by automated microscopy or unknown genital infections are identified manually.

Even when methods such as “isolation” or “cultivation” of bacteria is mentioned, you must know that this includes some type of microscope at the end, when the pathogens are identified.

Pathogens are most of the time invisible even under a microscope, and therefore the contrast needs to be increased against the background. The easiest method is staining with various agents such as non-specific or specific stains (e.g. gram staining, immunofluorescence).

Historic discovery and diagnosis of spirochetes

Borrelia are thin and weak, and therefore it is tedious to stain them with good efficiency. What is more, the thickness of Borrelia is below the resolution of many conventional light microscopes. This is why a microscope with a special illumination is used on these spirochetes. This device, the dark-field microscope was routinely used to study spirochetes in the 19^th century already, and it was with this method that the first ever disease was linked to a bacterial causative agent. Borrelia recurrentis, a close relative of Borrelia burgdorferi, was identified as causing relapsing fever. In 1873, Otto Obermeier published his observation that spirochetes could be identified in the patient’s blood by dark-field microscopy during recurrent febrile periods, but not in relatively asymptomatic periods.

Since 1909, examinations of unstained and unfixed preparations by dark-field microscopy have been able to show with complete certainty the presence of infection caused by Spirochetes, even before the appearance of antibodies (1). This is the point when dark-field microscopy entered the area of routine diagnostics.

It is important to mention that Borrelia burgdorferi was first identified by dark-field microscopy, and it is still routinely used in scientific experiments when Borrelia are isolated (2).

Finding Borrelia in a blood sample

Borrelia burgdorferi primarily spreads though the blood and it appears in remote body locations even a few hours after the start of the tick bite. (3)

The detection of Borrelia in the blood, however, poses a more difficult question. Borrelia are weak and their numbers decrease sharply after the blood collection, if not put under preferable conditions, i.e. in a special medium (2).

Furthermore, the blood cells in the sample will start to decompose, forming spirochete-like forms, so called pseudo-spirochetes or myeloid figures. (4); (5); (6); (7)

If the sample quantity is small, and the sample taking procedure is not clean enough, then the spirochetes normally present on the skin or in the environment may appear in the drop of blood investigated. This happens in case of “finger-prick” examinations. Thus, both pseudo-spirochetes and external contamination might cause a false positive result – there are many self-made scientists looking at a drop of blood and posting videos about finding Borrelia.

Is microscopy useless then in diagnosing Lyme borreliosis?

It has been said that the theoretical limit of microscopic investigations in blood is the number of spirochetes in the blood sample. (13)

It is known that the dynamics of the bacterial population becomes waving after a few days, but even the smallest concentration of Borrelia will be a million organisms per millilitre (8), albeit this is a hundred times smaller than the number of blood cells. Thus, the number of Borrelia is, at almost any point of time during the infection, sufficient to carry out a well-planned direct investigation. (3) It has been shown that Borrelia can be cultivated from blood samples (9), which confirms that the concentration must be sufficiently high for other investigations. (N.B. the amount of human genetic material present in the blood sample also outnumbers borrelia genome, which might block the use of PCR tests)

Despite the fact that there are a few publications about the successful use of dark-field microscopy in the diagnosis of Lyme borreliosis (10), the approach followed there is also questionable, as some opinions state the opposite (11); (12).

Either way, all the experiments carried out in the above publications used a small quantity, a drop of “finger-prick” blood, which is far from being enough, and under circumstances where the formation of pseudo-spirochetes, that is false positive borrelia, could not be closed out.

A proper solution

So far, the only method that has the theoretical and practical basis to be successful is DualDur.

DualDur® reagent and DD-LYME 4.0 ®:

• avoids the deterioration of Borrelia in the blood, by adding specific nutrients required
• stops the formation of pseudo-spirochetes by conserving the blood cells
• stops the motion of the human cell forms by stiffening the membranes
• provides enough sample for investigation and avoids external contamination by drawing 4ml of venous blood into a closed syringe
• concentrates the scarce spirochetes from the sample so there is always a sufficient number, at every stage of the infection
• avoids human error by standardising the sample taking and preparation
• reduces investigator error by automating the investigation process
• provides statistics of the sample by automatically scanning the whole area
• is repeatable because the process is automated and standardised
• is clinically tested to provide a 96% Positive Predictive Value
• CE marked for use across the EU

References

1. Spirochaeta pallida: Methods of examination and detection, especially by means of the dark-ground illumination. 1909., Brit. med. J., pp.: 1117-20.
2. Isolation and Characterization of Borrelia burgdorferi Sensu Lato Strains in an Area of Italy Where Lyme Borreliosis Is Endemic, Ciceroni et al, J Clin Microbiol. 2001 Jun; pp: 39(6): 2254–2260.
3. Population Dynamics of Borrelia burgdorferi in Lyme Disease. S. C. Binder, A. Telschow, M. Meyer-Hermann, Front Microbiol. 2012; 3: 104.
4. A new spirochaeta found in human blood. Chamber, H. 1913., Lancet, pp.: 1: 1728–1729.
5. Present status of spiculed red cells and their relationship to the discocyte, echinocyte transformation: a critical review. Brecher, G., Bessis, M. 1972., Blood, pp.: 40: 333–344.
6. : Pseudospirochetes a cause of erroneous diagnoses of leptospirosis. . Smith, TF., et al. 1979., Am. J. Clin. Pathol., pp.: 72: 459–63.
7. Pseudospirochetes in animal blood being cultured for Borrelia burgdorferi. Greene, RT., Walker, RL. , Greene, CE. 1991., J. Vet. Diagn. Invest., pp.: Oct; 3(4): 350–2.
8. Brief communication: hematogenous dissemination in early Lyme disease. Wormser G., McKenna D., Carlin J., Nadelman R., Cavaliere L., Holmgren D., Byrne D., Nowakowski J. Ann. Intern. Med. 2005: 142, pp. 751–755
9. Isolation of Borrelia burgdorferi from the blood of seven patients with Lyme disease, Nadelman RB et al., Am J Med., pp. 88(1):21-6., 1990 Jan.
10. A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques, M. Laane, I. Mysterud, 2013, Biology
11. Microscopy of human blood for Borrelia burgdorferi and Babesia without clinical or scientific rationale, Dessau R. B., INFECTIOUS DISEASES, 2016, EDITORIAL COMMENTARY.
12. Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and detecting Borrelia and Babesia organisms in human blood., Aase A et al., Infect Dis (Lond). , pp. 48(6):411-9. , 2016;.
13. Borrelia: Molecular Biology, Host Interaction and Pathogenesis, Samuels DS, Radolf JD, Caister Academic Press; 2010, 547. ISBN 978-1-904455-58-5