Blood cell disruption to significantly improve the Borrelia PCR detection sensitivity in borreliosis in humans
Introduction
Lyme disease is caused by a spirochete belonging to the Borrelia burgdorferi sensu lato complex (B. burgdorferi), which may harbour multiple and various clinical symptoms including arthritis, meningo-encephalitis as well as non-specific polymorphic signs. The latters include asthenia, neuropathic pain, dizziness, tremor, weakness, cramps, muscle fasciculations, dyspnea, swelling, and joint and muscle pain [1]. Indeed, Lyme disease can feature a confusing multisystemic infectious disease comprising a variety of misleading clinical symptomatologies.
The final diagnosis is usually confirmed by using serological tests (ELISA followed by Western Blot, part of the classical two-tier Lyme disease testing). However, the serology sensitivity ranges from 34% to 70.5%, and many infected patients still remain with false-negative diagnosis tests and with no curative treatment [2], [3], [4], [5], [6], [7].
This huge gap could be explained by multiple serotype variants of Borrelia species causing Lyme disease. This also could be the result of the use of large spectrum of antibiotics strategies, as the sequestration of anti-Borrelia antibodies within immune complexes, and/or the intracellular location of the cystic form presenting with quiescent metabolism have been reported [8], [9], [10]. Furthermore, in patients having experienced a chronic form of the disease, the IFN-gamma response is not followed by an increase of IL-4 expression. In such patients this suggests the persistence of the Th1 response, and the deficiency in the Th2 response, the subsequent lack of humoral response resulting in the false-negative results of the patient’s serology [11], [12]. Regarding this confusing disease, the keypoint is that some patients infected by Borrelia have no final diagnosis and will not be definitely cured. The reasons why include first, that Borrelia could indirectly enhance mechanisms of an autoimmune disease; second, that ticks can transmit many other bacteria, virus and parasites (including Bartonella, Ehrlichia, Anaplasma, Babesia for example) than the B. burgdorferi. Indeed, alternative diagnostic techniques such as polymerase chain reaction (PCR) or Enzyme-Linked Immunospot (ELISPOT) serum analysis of infected patients could be helpful to reach the final diagnosis, which obviously can fail due to the lack of its sensitivity [13], [14], [15]. According to our own experience, some false-negative test- patients indeed exhibit a significant clinical improvement while receiving proper antibiotics’ therapy.
To enhance the sensitivity of PCR detection of the bacteria, we promote to increase the detection sensitivity of the diagnostic test; first by using concentration methods of pathogen sample pre-enriching ; second by increasing the number of released bacteries available by lysing the infected patient’s peripheral blood mononuclear cells (PBMC) that contain the masked intracellular bacteria.
Several studies have reported that B. burgdorferi is able to infect in vitro and in vivo immune cells as well as fibroblasts, keratinocytes, endothelial, neuronal and glial cells [16], [17], [8], [18]. Consequently, cell membrane lysis techniques will unmask and release bacteria into the patient’s blood, thus increasing the Borrelia target and their subsequent detection.
Section snippets
Hypothesis
It is well established that main causative agents of Lyme syndrome are bacterial spirochetes from the Borrelia genus transmitted by ticks during their blood meal. As infected ticks feed on a subsequent host, Borrelia exit the gut, invade the salivary glands and, along with tick saliva, transmit to the human’s skin during the tick’s bite. Subsequently, these spirochetes migrate and disseminate through blood from the endoderm to organs, including joints, heart and even the central nervous system.
The hypothesis testing
In patients suffering from Lyme syndrome due to Borrelia, bacteria circulating in the patient’s blood as well as intracellular bacteria are few. The diagnosis made by molecular methods (end point PCR, quantitative PCR, High-throughput sequencing…) becomes thus highly challenging since techniques need to reach the bacterial nucleic targets. Those Bacteria are surrounded by a stiffer cell wall than the soft human cell membranes. Indeed, the strong bacterial wall is far more resistant to lysis
Consequences of the hypothesis and discussion
In our experience, diagnosis of Lyme disease due to Borrelia is difficult in some cases, where the clinical presentation is polymorphic and when the currently performed above-mentioned tests have a low efficiency [2], [3], [4], [5], [6], [7]. We assume that the bacterial DNA target could be reachable; therefore, the blood PCR testing could play a pivotal role to get a reliable diagnosis of the Borrelia-induced Lyme syndrome in patients presenting with a false negative serology. Furthermore,
Conclusion
Blood cell disruption technique could increase the detection of Borrelia in the patient’s blood samples. Therefore, this may contribute to enhance the PCR sensitivity, leading to full diagnosis of borreliosis in infected patients who present with equivocal clinical features. In addition, biofilm and cell disruption technique/test approach could be useful to improve the detection of other co-infecting bacteria transmitted by ticks. This should lead to administer the proper antibiotic therapy and
Conflict of interest
None.
Competing interests
None.
Authors’ contributions
Alexis Lacout: promoted the design of the article; Michel Franck: participated in the design of the article; Pierre Yves Marcy: participated in the design of the article; writing assistance; Marie Mas: writing assistance; Christian Perronne: participated in the design of the article; important intellectual content; Yves Moné: participated in the design of the article; writing assistance; Francisco Veas; deep writing and conceptualization.
All authors read and approved the final manuscript.
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These authors contributed equally to this work.