Shiva Kumar Goud Gadila1, Gorazd Rosoklija2,3, Andrew J. Dwork2,3,4,5, Brian A. Fallon2* and Monica E. Embers1*
10 May 2021
https://www.frontiersin.org/articles/10 ... 28045/full
The complex etiology of neurodegenerative disease has prompted studies on multiple mechanisms including genetic predisposition, brain biochemistry, immunological responses, and microbial insult. In particular, Lyme disease is often associated with neurocognitive impairment with variable manifestations between patients. We sought to develop methods to reliably detect Borrelia burgdorferi, the spirochete bacteria responsible for Lyme disease, in autopsy specimens of patients with a history of neurocognitive disease. In this report, we describe the use of multiple molecular detection techniques for this pathogen and its application to a case study of a Lyme disease patient. The patient had a history of Lyme disease, was treated with antibiotics, and years later developed chronic symptoms including dementia. The patient's pathology and clinical case description was consistent with Lewy body dementia. B. burgdorferi was identified by PCR in several CNS tissues and by immunofluorescent staining in the spinal cord. These studies offer proof of the principle that persistent infection with the Lyme disease spirochete may have lingering consequences on the CNS.
Neuroborreliosis can occur in up to 15% of patients with Lyme disease, affecting both the central nervous system (CNS) and peripheral nervous system (PNS). The disease of the nervous system can become chronic and debilitating. Prior studies of persistent post-treatment Lyme encephalopathy demonstrated both immune activation in CSF and serum and metabolic and blood flow deficits in the CNS (1–3). While the persistence of the pathogen after antibiotic treatment in humans remains controversial, animal studies have clearly demonstrated its occurrence (4–8). Evidence from experiments performed in mice, dogs and primates have shown that intact spirochetes can persist in the mammalian host after the administration of antimicrobial drugs, and that they can be metabolically viable (9). Studies in vitro have demonstrated that persister Borrelia develop stochastically in the presence of microbiostatic antibiotics and that tolerance is enabled by slowed growth (10, 11).
We have recently demonstrated both inflammation and persistence of Borrelia in the CNS and PNS of doxycycline-treated rhesus macaques that were infected with the Lyme disease pathogen (9, 12). In humans, persistence has been studied early after treatment and in Post-Treatment Lyme Disease (PTLD) patients. In one study, skin biopsies were taken from the erythema migrans (EM) lesion and after treatment (~2 mo later). Approximately 1.7% of these were culture-positive and confirmed as the same strain (13, 14). Human xenodiagnoses were also performed in a more recent study. Larval ticks were placed on patients who had EM (early stage) or PTLDS (15). Tick samples were evaluated by PCR and culture; of the 23 patients on whom ticks fed and were recovered, 19 were negative, 2 were indeterminate, and 2 were positive by PCR (1 patient with EM and 1 with PTLDS). Two other studies have indicated that the spirochetes could be cultured from late stage Lyme patients, yet the cultures took many weeks and rounds of subculturing without active growth (16, 17). Thus, in the absence of a reliable detection system, persistent infection in humans remains difficult to assess. One means to address this issue is to interrogate patient tissue for persistent pathogen through the analysis of post-mortem specimens.
In this report, we describe the use of multiple overlapping techniques, including immunofluorescence assay (IFA), RNA in situ hybridization (RNAscope), and PCR for detection of Borrelia spirochetes in post-mortem tissues. As example, we describe the detection of B. burgdorferi in the brain tissue of a post-mortem donor from the brain repository of the Lyme and Tick-Borne Diseases Research Center at the Columbia University Irving Medical Center. This individual had a history of Lyme disease that appeared to have been successfully treated with antibiotics; 4 years later developed a neurodegenerative disorder leading to dementia.
Case Study Description
This 69 year old woman (Patient 12,577) contracted Lyme disease at age 54 with a well-documented erythema migrans rash accompanied by a severe headache, joint pains and a fever of 104; convalescent serologies were positive on ELISA and both IgM and IgG Western blots. Treatment with doxycycline for 10 days led to symptom resolution. Two years later, a sleep behavior disorder emerged. Four years later, cognitive problems (processing speed, mental tracking, and word-finding) emerged and gradually worsened. Other symptoms included photophobia, paresthesias, fasciculations, and myoclonic jerks. Neurocognitive testing revealed deficits in visuospatial skills and executive functions with preservation of verbal skills, suggesting a neurodegenerative process. Brain Magnetic Resonance Imaging with and without contrast showed mild atrophy and non-specific scattered white matter hyperintensities without enhancement. Brain Single Photon Emission Computed Tomography scans showed decreased perfusion in the right posterior parietal and temporal lobes. Serum was negative or normal for erythrocyte sedimentation rate, c-reactive protein, antinuclear antibody, and thyroid stimulating hormone. PCR assays of blood for Bartonella henselae, Babesia microti, and Borrelia burgdorferi were negative. Serum C6 ELISA was negative but Lyme IgG Western blot was positive with 9/10 bands. Treatment with IV ceftriaxone at age 60 for 8 weeks led to 60% improvement in cognition and interpersonal engagement; oral amoxicillin 500 mg three times daily was continued for 6 months after the IV treatment. The initial improvement was not sustained and subsequent antibiotic therapy with minocycline was of no clear benefit; gradually her visual spatial skills and executive functions deteriorated further, and anxiety worsened. Serum IgG Western blot continued to be positive. At age 62, a cerebrospinal fluid study demonstrated 4 CSF IgG bands on Lyme Western blot; unfortunately, because CSF and serum ELISA studies were not conducted, intrathecal Bb specific antibody production could not be assessed. Other CSF studies were unremarkable including absence of pleocytosis or elevated protein, absence of P-tau elevation, Venereal Disease Research Laboratory assay, Acid-Fast bacteria, fungi, and negative Herpes Simplex Virus and Epstein-Barr Virus PCRs. A second brain MRI showed periventricular and subcortical T2 hyperintensities possibly due to “small vessel ischemia or demyelinating disorders like Lyme disease.” Fluorodeoxyglucose-Positron Emission Tomography scan showed “diffuse cortical hypometabolism, worse in the posterior parietal and temporal lobes, with sparing of the sensory motor cortex and visual cortex bilaterally—findings consistent with Alzheimer's disease.” The extensive workup at that time led to the diagnoses of both a REM behavioral disorder with verbalizations and movements and a neurodegenerative dementia characterized by expressive aphasia, visual agnosia, anomia, deficits in executive function and calculation, and mild memory problems. Eventually, she developed severe oral dystonia, making feeding progressively more difficult; she died 15 years after the initial infection with B. burgdorferi. Early and severe movement disorders, REM behavioral disorder, paranoia, and personality changes all favored a clinical diagnosis of dementia with Lewy bodies.
Two reasons exist for the interrogation of autopsy specimens for the Lyme disease spirochete. First, in patients with a known history of Lyme disease and a record of antibiotic treatment, the potential for treatment to fail in eradicating the infection can be evaluated. Notably, a detailed patient history, including history of possible second B. burgdorferi infection and treatment non-compliance, is necessary. Given the difficulty in recovering organisms from living people, looking at post-mortem tissue can provide some resolution on the issue of persistence. Secondly, patients such as the one presented here, can manifest neurological disease that may or may not be related to infection. Here, the patient developed dementia with Lewy body pathology. While availability of tissue may be a challenge, the role of Borrelia burgdorferi in the etiology of chronic neurological disease, can be studied as a “proof of principle.”
However, there are studies showing the expression of OspA in one-third of the spirochetes inoculated in mice and in cerebrospinal fluid of early neurologic Lyme disease (52, 53), suggesting that OspA might not be an ideal choice in the interpretation of the analysis of a study
The gene expression profile of long-term persisters within a host is as yet unknown.