Spirochetes were discovered by the Dutch Anthonij van Leeuwenhoek by microscopic tests as early as 1683 and he reported his discovery to the Royal Society.  Two centuries later József Fodor was the first to realise that the blood of healthy living organisms did not contain microbes. Even today medical professionals consider that there is an advantage of the morphological – microscopic – method, in comparison to serological and PCR tests, in that its result does not depend on various type changes – primarily characteristic of Borrelias among the Spirochetes – or the appearance of new sub-strains. During the course of the disease, a genetic and/or phenotype modification of the pathogen may develop in the patient (14) (15) (16) that might not be detected with the currently existing procedures. Being able to identify a specific morphology in a sample can be an evidence of the infection, as a qualitative investigation.

Ever since 1909 it has been possible to identify spirochetes with certainty using dark-field microscopy(17). These tests using unstained, unfixed preparations were effective because the samples were taken from the primary chancre infected with Treponema.

The laboratory diagnosis of Borreliosis causing a wide range of clinical symptoms can be based on the detection of Spirochetes from the blood. It is especially easy in the case of Febris recurrens, because Borrelia recurrentis usually appears in larger numbers, and the easy staining of the causative agent also simplifies microscopic detection. There are, however, mild cases when, due to the low cell count, the clinical suspicion cannot be confirmed using this test. To solve this problem, the microhematocrit concentration method has been used since 1972. During this procedure, the sample is concentrated by double centrifugation of the blood sample. Concentration of the small number of pathogens has been a routine procedure in parasitology to increase the diagnostic yield of the classical microscopic method. This is a more reliable method of detecting scarce pathogens, instead of directly investigating the sample.

In practice, the diagnosis of Lyme borreliosis including the identification of its causative agent poses a significant problem.  After unsuccessful tests performed with state-of-the-art devices, Willy Burgdorfer was able to identify the causative agent of Lyme borreliosis as an unknown Spirochete by a simple microscopic test. This pathogen is currently referred to as Borrelia burgdorferi sensu lato, a collective name (18). As mentioned before, morphological tests have traditionally been part of the laboratory diagnosis of spirochetosis. When concentrated native preparations of body fluids are examined with dark-field microscopic tests, it is not necessary to use staining, so the extraordinarily thin and long Spirochetes do not get washed off the slide during the steps of staining; hence, the sensitivity of the test improves significantly. However, pseudospirochetes, also called myeloid figures, which are primarily produced during the decomposition of red blood cells present both in animal and in human samples, can easily mislead the investigator. This artefact appears especially frequently when stored blood samples or the intestinal contents of blood-sucking insects – fleas and ticks – are evaluated for infection. The above-mentioned facts are covered by the following literary sources: (19); (20); (21); (22);

It is also known that the Borrelia burgdorferi sensu lato shows an extraordinarily diverse morphology, even in cultures. Under the influence of antibiotics or other environmental effects, degenerative figures may appear. The formation of such artefacts must also be avoided in experimental tests.

References

14. Association of genetic variability within the Borrelia burgdorferi sensu lato with the ecology, epidemiology of Lyme borreliosis in Europe. Derdáková M, Lencáková D. 2002., Ann Agric Environ Med, pp.: 12(2):165-72.
15. Global analysis of Borrelia burgdorferi genes regulated by mammalian host-specific signals. Brooks CS, Hefty PS, Jolliff SE, Akins DR. 2003., Infect Immun., pp.: 71(6):3371-83.
16. Regulation and expression of bba66 encoding an immunogenic infection-associated lipoprotein in Borrelia burgdorferi. Clifton DR, Nolder CL, Hughes JL, Nowalk AJ, Carroll JA. 2006., Mol Microbiol., old.: 243-58.
17. Spirochaeta pallida: Methods of examination and detection, especially by means of the dark-ground illumination. 1909., Brit. med. J., , pp.: 1117-20
18. Lyme Disease – a tick-borne spirochetosis? Burgdorfer, W, Barbour, A. G., Hayes, S., F., Benach, J. L, Grunwaldt, E., Davis, J. P. 1982. June 18., Science, pp.: 216 (4552): 1317–9.
19. A new spirochaeta found in human blood. Chamber, H. 1913., Lancet, p.: 1: 1728–1729
20. Present status of spiculed red cells and their relationship to the discocyte, echinocyte transformation: a critical review. Brecher, G. és Bessis, M. 1972., Blood, pp.: 40 333–344.
21. Pseudospirochetes a cause of erroneous diagnoses of leptospirosis. Smith, TF., et al. 1979., Am. J. Clin. Pathol., pp.: 72 459–63.
22. Pseudospirochetes in animal blood being cultured for Borrelia burgdorferi. Greene, RT., Walker, RL. and Greene, CE. 1991., J. Vet. Diagn. Invest., pp.: Oct; 3(4): 350–2.