Review and rebuttal of the article

„Is There a Role for Dark Field Microscopy in the Diagnosis of Lyme Disease? A Narrative Review” by Uğur Önal , Fatma Saraç-Pektaş, İmran Sağlık

Rebuttal prepared by the team of Lyme Diagnostics Ltd, led by András Pál Bózsik

Original publication available: https://pmc.ncbi.nlm.nih.gov/articles/PMC10986710/

 

Summary

The above review article does not aim to be a systematic one, and it almost certainly reflects the attitude and opinion of many scientists at the time of writing.

Nevertheless, it falls short in terms of scientific evidence that is based on facts rather than on literature references, which in turn are based on opinions and outdated views.

Its aim was to provide guidance for Turkish doctors who are puzzled and sometimes misled by scientists. These scientists identify Borrelia from unreliable samples using untested methods, in good faith, but sometimes without proper knowledge of Borrelia research. Still, the above review misses its objective and targets a method that has been used for centuries to detect various causative agents: microscopy.

The review uses “factual references” to the literature, which can be grouped around three topics:

  • The scarcity of Borrelia in patient samples – which is true in itself but is now addressed by culturing and/or concentrating
  • The presence of artefacts, especially in a single publication whose clinical utility is rightly doubted, does not exclude the possibility that other methods can avoid creating artefacts (e.g. culturing, specific staining, or using special blood-draw liquids containing specific additives)
  • Long-standing beliefs that were never supported by facts, but at best by consensus or by outdated literature that, in turn, contains no facts either.

 

Discussion

Key points that require fact checking and correction

  1. False information, no support for statement from cited literature

“Dark field microscopy is a diagnostic technique that can be used for the detection of various microorganisms; however, imaging techniques, such as immunofluorescence staining or cell sorting of cell wall-deficient or cystic forms of Borrelia burgdorferi, are not currently recommended for the diagnosis of Lyme disease because of certain limitations (7). (A)”

a. Immunofluorescence is, in some cases, funded by the German health insurance system.

b. The citation does not support the statement:

The link leads to the USA CDC website, where only the single recommended test is featured. The non-recommended tests are not featured. At the same time, the CDC itself states the limitations of the so-called Modified Two-Tier Test (MTTT), which makes it incapable of detecting many stages of the infection. Additionally, the disease differs in North America (where most tests were developed) from that in Europe. For this reason, the EU Parliament issued a resolution in 2018 stating that the standard of care in diagnosis is clinical, and that country guidelines should be modified to reflect that. (B) The resolution was passed in November 2018; the current article was published in December 2023. By the time the publication appeared, the national guidelines had not been modified to reflect the resolution.

 

  1. No support for statement from cited literature

The article quotes another publication

“According to the European Society of Clinical Microbiology and Infectious Disease (ESCMID) Study Group on Lyme borreliosis (ESGBOR), molecular methods can be used for the detection of Borrelia as supplementary diagnostic methods for particular indications, and the visual contrast sensitivity test cannot be recommended for diagnosis due to low specificity (9).” (C)

(from the article itself)

“Several diagnostic tests, such as the visual contrast sensitivity test, the lymphocyte transformation test, and CD57+/ CD3- lymphocyte subpopulation typing, cannot be recommended for diagnosis of LB because they lack specificity [60,71,98].” (D,E,F)

(from the quoted publication (C))

The phrase “visual contrast sensitivity test”, or “microscopy” does not even appear in any of the cited literature 60,71,98 (D,E,F). There is no reference to visual methods whatsoever in any of the 3 cited articles!

At the same time, “A visual contrast sensitivity (VCS) test measures your ability to distinguish fine details at low contrast levels, such as faint shades of grey against a background.” It is an eye and neurology test and is by no means an alternative expression for dark-field microscopy.

 

  1. No support for statement from cited literature

“Dark-field or phase-contrast microscopy is not recommended for Lyme borreliosis because of a lack of sensitivity and specificity in guidelines from the French scientific societies and CDC (7, 10, 11).” (A,G,H)

Reviewing the references one by one:

a. 7: As mentioned above, the cited CDC guidelines (7) (A) do not contain any recommendations against any method.

b. 10: The French guidelines (10) (G) quote another article,

“Microscopy can be used to interpret culture results, but direct microscopy on samples is not recommended because of its lack of specificity. Histology is useful for the diagnosis of ACA and for differential diagnoses, but the result is not indicative of active Lyme borreliosis [21].” (I)

The latter reference (21) (I) only contains diagnostic guidelines for ACA, but does not mention microscopy!

c. 11: The position paper of ESGBOR, the ESCMID study group for Lyme borreliosis (11) (H) references three publications, of which two are about microscopy, and both encompass the rebuttal of the same method. The third reference is not about microscopy.

“Tests such as CD57 cell count and dark-field or phase-contrast microscopy have no rationale and have been found to be non-specific [82-84].” (J,K,L)

The first two references are dealt with at the website of lymediagnostics.com

https://lymediagnostics.com/2021/01/11/what-is-wrong-with-microscopy-in-borreliosis/ (M)

They are the scientific investigations and rebuttals of the publication: “A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques” by M. Laane and I. Mysterud (N). This will be reviewed in the next paragraph.

 

  1. The method of Laane, and I. Mysterud. (N)

This method is rightly criticised for the methodological errors, namely:

  • Sample collection issues
  • Showing known artefacts instead of Borrelia
  • No tested cutoff level for a diagnostic purpose

Hence, the conclusion that this method is not valid for the laboratory diagnosis of Lyme borreliosis is valid. This is a 2013 publication that has been methodologically and correctly criticised in an article and a commentary in 2016 (J,K).

However, the DualDur diagnostic method, based on automated microscopy, has been clinically proven to detect Borrelia and to have a clinically viable diagnostic cutoff level, and the presence of Borrelia has been demonstrated in two publications (2024 and 2025) (O,P). Therefore, information from a 2016 article cannot be generalised any more to a diagnostic test developed after that publication, nor to any other methods that have not been tested and disproven.

  1. Outdated/limited information

A previous systematic review of ‘direct microscopy of human tissues’ emphasized that the modified dark-field microscopy technique should not be used for diagnosis and Borrelia detection by microscopy can only be used for research purposes (12). (Q)

The review (12) (Q) from 2019, quoted here, stated that microscopy was “research purpose only”. This statement was superseded in Europe by the issue of the IVD CE registration document for DualDur automated microscopy in February 2020. DualDur became the first CE IVD-registered direct diagnostic method in Europe, using automated microscopy and artificial intelligence to detect Borrelia by thoroughly scanning the sample and calculating statistics from it. The clinical trial also confirmed previous findings that manual dark-field microscopy can detect Borrelia in a sample, but human investigators are unable to keep track of the statistics required for the diagnostic cutoff level.

 

  1. Opinion based on consensus, not data

“GDS (17) (R) Direct detection of Borrelia in patient samples using light microscopy is currently not recommended.”

The reference (17) (R) is based on a 2017 guideline, which states:

“Direct detection of Borrelia in patient samples using light microscopy is currently not recommended. (Strong consensus: 18/19)”

The consensus is already outdated for the same reason as Paragraph 5, and it is not based on facts and data but on consensus.

 

  1. Facts quoted without numbers, can be accepted based on personal experience

“Patients in Turkey often receive non-recommended tests (such as dark field microscopy and lymphocyte transformation tests) during the diagnostic process because recommended diagnostic methods are not available in many centers.”

In our experience, some microscopists in Turkey use methods similar to those of Laane and Mysterud, which are generally based on examining a drop of blood under manual dark-field microscopy. The sample is not protected from superinfection from the skin, from the production of artefacts, or from the decomposition of Borrelia, and sometimes unrealistic morphologies are identified as Borrelia.

  1. Contradicting and outdated information

“Branda et al. reviewed the laboratory diagnosis of Lyme disease and emphasized that direct visualization of Borreliae in blood or other infected tissues easily leads to misinterpretation. Direct visual detection is less sensitive or practical than a first-line diagnostic or adjunctive test because of the low in vivo organism burden of primary tissue samples (22).” (S)

The relevant part of the reference 22 (S) is

“Direct microscopic visualization.

In contrast with relapsing fever borreliae, direct visualization of Lyme-related borreliae in blood or other infected tissues is very challenging and easily subject to misinterpretation. The in vivo organism burden is usually so low in primary tissue samples that direct visual detection is not sensitive or practical as a first-line diagnostic approach … (255).

And then it goes on to cite all the staining methods and culturing techniques that still make it possible to view Borrelia in primary samples. The information is partially correct, because the Borrelia burgdorferi sensu lato group causes a bacterial burden that is 2-3 orders of magnitude lower than that of relapsing fever type Borrelia, and therefore concentration or culturing is required to reach a proper limit of detection (O).

The reference then goes on to explain that

“In combination with serum antibody testing, microscopic tissue examination for B. burgdorferi spirochetes may serve an adjunctive diagnostic role, but direct examination requires considerable experience.”, thus the method can be used, but requires specific knowledge.

 

  1. Outdated information

“Lohr et al. also reviewed the diagnostic utility of direct microscopy for Lyme disease and mentioned the limited clinical utility because of the sparseness of organisms in samples (23).” (T)

The 2018 reference 23 (T) states that

“Direct microscopic detection of B. burgdorferi s.l. has limited clinical utility in the laboratory confirmation of LB due to the sparseness of organisms in clinical samples [2,3,56–58].”

Whereas the scarcity of Borrelia in clinical samples has already been disproven by several methos including PCR and culture (O,P).

 

  1. Conclusion stating the obvious and the requirements for an acceptable microscopic method – at the same time not allowing that some methods or scientists could achieve the required expertise

“The literature clearly shows that the sensitivity of dark field microscopy is low, and it may produce false negative and positive results. In addition, factors such as the technician’s expertise and the specimen’s quality should also be considered. Therefore, direct detection of Borrelia from patient material using dark field or focus floating microscopy is not recommended for diagnostic purposes (28).” (U)

The sampling, preparation, conservation and concentration methods, and the statistics used during the examination, truly influence the quality of the examination and its reliability for a diagnosis. In agreement with the above statement, the article also outlines the failure points of the current methods, and we can easily provide methods to avoid these issues:

  • Haemoculture sampling of 4 ml of venous blood can avoid superinfection from a single drop of blood obtained by finger prick
  • Special blood draw or culture medium can prevent deterioration of spirochetes
  • Concentration or culturing can increase the scarce number of Borrelia in the blood
  • Special additives can prevent artefacts such as pseudospirochetes
  • Automated scanning of the sample can avoid human error and reduce the need for an expert at the sample scanning stage
  • Statistics collected during automated microscopy can provide a diagnostically valid cutoff
  • Central expert validation of the statistics and Borrelia can help avoid human error

All the above solutions are provided by DualDur, which was IVD CE registered in 2020, based on the results of the largest prospective clinical trial in Europe. Two peer-reviewed scientific publications attest to the method’s scientific value (O, P).

 

Note: in this rebuttal, the original numbering of the cited literature is used; therefore, the reference list is provided with alphabetical marking.

References

A. Lyme Disease: Laboratory tests and practices that are currently recommended. Atlanta: Centers for Disease Control and Prevention (CDC) [November 8, 2023]. https://www.cdc.gov/lyme/diagnosistesting/labtest/otherlab/index.html
B. European Parliament resolution of 15 November 2018 on Lyme disease (Borreliosis) (2018/2774(RSP)) https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=oj:JOC_2020_363_R_0014
C. Stanek G, Fingerle V, Hunfeld KP, Jaulhac B, Kaiser R, Krause A, et al. Lyme borreliosis: clinical case definitions for diagnosis and management in Europe. Clin Microbiol Infect. 2011;17(1):69–79. doi: 10.1111/j.1469-0691.2010.03175.x.
D. Marques A Chronic Lyme Disease: A Review Infectious Disease Clinics, 22, 341-360
E. Wilske, B. ∙ Fingerle, V. ∙ Schulte-Spechtel, U. Microbiological and serological diagnosis of Lyme borreliosis FEMS Immunol Med Microbiol. 2007; 49(IV):13-21
F. Marques, A. ∙ Brown, MR ∙ Fleisher, TA Natural killer cell counts are not different between patients with post-Lyme disease syndrome and controls Clin Vaccine Immunol. 2009; 16(II):1249-1250
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I. Skin Manifestations of Lyme Borreliosis; Diagnosis and Management; Therapy in Practice; Diagnosis and Management of Cutaneous Lyme Borreliosis; Published: 10 September 2012; Volume 9, pages 355–368 (2008)
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M. https://lymediagnostics.com/2021/01/11/what-is-wrong-with-microscopy-in-borreliosis/
N. A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques, M. Laane, I. Mysterud, 2013, Biology
O. Bózsik, A.P.; Bózsik, B.P. First Application of an Improved-Resolution Dark-Field Microscopy Setup, Combined with a Novel Direct-Staining Live Immunofluorescence Investigation Method, Visualising the Motion Of Borrelia Burgdorferi Spirochetes. Acta Microsc. 2024, 33, 54.
P. Bózsik, A.P.; Déri, J.; Bózsik, B.P.; Egri, B. Presence of Borrelia Spirochetes in White Stork (Ciconia ciconia), White-Tailed Eagle (Haliaeetus albicilla), and Eastern Imperial Eagle (Aquila heliaca): Hospitalized in a Wild Bird Hospital and Sanctuary (Hortobágy, Hungary). Animals 2024, 14, 3553.
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