https://www.frontiersin.org/articles/10.3389/fneur.2021.628045/full Go here for full study. Excerpts below:
Detecting Borrelia Spirochetes: A Case Study With Validation Among Autopsy Specimens
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 vitrohave 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. burgdorferiin 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.
Human Control Tissues
Tissue blocks from various regions of seven specimens from brains of deceased Macedonian residents that were housed in the Macedonian/New York State Psychiatric Institute Brain Collection were used as controls. Though none had a clinical history of Lyme disease based on interview with the surviving family members, Borrelia is endemic in Macedonia. These brain tissues were probed in the same manner as the human case study with IFA and PCR-based detection methods.
The Case Study Pathology Is Characteristic of Dementia With Lewy Bodies (DLB)
The fresh brain weighed 996 g and appeared atrophic Coronal sections through the left cerebral hemisphere and brain stem revealed mild enlargement of the lateral ventricle, particularly the temporal horn. The substantia nigra was normally pigmented or nearly so. Microscopically, nigral and cortical Lewy bodies, were seen with hematoxylin and eosin stain (H&E, Figures 4A,B). Immunohistochemistry (IHC) for α-synuclein (clone 42, BD Transduction Laboratories) showed numerous immunoreactive Lewy bodies and fibers in substantia nigra, hippocampal formation and neocortex, Figures 4C–E). IHC for hyperphosphorylated tau (monoclonal antibody AT8; ThermoFisher) revealed intense staining of many limbic neurofibrillary tangles and neuropil threads (Braak stage 2–3, Figure 5), and of occasional neurofibrillary tangles in neocortex, but senile plaques were extremely rare, and each contained only a few fibrils (Figure 5). H&E showed prominent thickening of small blood vessels in gray and white matter, extensive mineralization of pallidal vessels, and rare microglial nodules in the hippocampal formation. Immunohistochemistry for Iba-1 (Wako), CD68 (clone KP1, Dako), and CD163 (clone EDHu-1; Bio-Rad) showed moderate numbers of activated microglia and large numbers of macrophages in hippocampal formation and spinal cord (Figure 6). In summary, we see DLB accompanied by features of Alzheimer’s disease, a common presentation.
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. burgdorferiinfection 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 burgdorferiin the etiology of chronic neurological disease, can be studied as a “proof of principle.”
Our study confirms that Borrelia burgdorferi was detected in the brain and spinal cord tissue of this patient with a history of previously treated Lyme disease. These results however do not clarify whether the Borrelia infection had anything to do with her progressive neurodegenerative disorder. It is possible this is an unrelated incidental finding or that there is a relationship between CNS infection with Bb and the development of a neurodegenerative dementing disorder.
Previous studies suggest that Borrelial spirochetes can start invading the nervous system during early stages of the infection resulting in meningeal seeding (29), and this later leads to neuroborreliosis. To define the pivotal neurological deficits, a study in Europe examined the clinical manifestations of 68 patients hospitalized for neuroborreliosis. Meningitis was found to be one of the least frequent conditions, present in 6% of the patients (30), whereas cranial neuritis was the most frequent (25%). The clinical Lyme case presented here was documented with meningismus at the time of the EM rash, supporting the possibility of mild meningitis at early infection. Bacterial meningitis leading to cognitive impairment was well-studied in Treponema pallidum in relation to dementia (31). B. burgdorferi infection has also been associated with mild (32) to severe (33) cognitive deficits. In the endemic areas of Lyme disease, Borrelia infections as a possible cause of cognitive impairment has to be carefully considered.
Neurotropic viruses have been associated with neurodegenerative syndromes, as have spirochetal infections (34–38). Precedence for an association between B. burgdorferi infection, specifically, and dementia exist (38–42), however there are also reports that have failed to link B. burgdorferi to AD (43). Evidence that amyloid plaques may have a functional protective role in combatting microbial infection has also come to the fore (44). Evidence that Borrelia can induce amyloid production is suggestive of a possible mechanism for development of AD (45–47).
To comprehensively evaluate the possible role of Borrelia in dementia (Alzheimer’s and LB), 20 patients were identified from a total of 1,594 patients who were seen for dementia, who had positive intrathecal anti-Borrelia antibody index (AI), indicative of past or present Lyme disease (48). Among these 20 patients, 7 patients with neuroborreliosis dementia showed stability or mild improvement in their cognitive functions after treatment with ceftriaxone, and the others showed progressive worsening despite antibiotic treatment (48). The individual in our clinical case reported 60% cognitive improvement after the antibiotic treatment. However, this improvement was not sustained and cognition gradually worsened, a finding consistent with a previous study demonstrating cognitive functional deficits in treated Lyme neuroborreliosis patients (49). The possible anti-inflammatory effects of antibiotic cannot be discounted (50).
A recent study aimed at testing the hypothesis that polymicrobial infections contribute to Alzheimer’s disease was conducted. Brain sample tissues were probed for B. burgdorferi using a commercially available monoclonal antibody (43). However, this study was unable to demonstrate the presence of Borrelia spirochetes in the tissue samples. The possibility exists that this could be due to the selection of antibody. The polyclonal used exhibited some cross-reactivity to fungal structures and the monoclonal antibody may have targeted an antigen (OspA) that is downregulated as spirochetes migrate from tick to mammalian host. Studies have shown that the expression of the OspA is abundant on the surface of bacteria when residing in tick midguts, but its expression is repressed during host infections (51). 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. In a recent study from our laboratory, we were able to identify B. burgdorferi with a monoclonal antibody to OspA in some tissues (e.g., heart) but not others, where they were positively identified with polyclonal antibodies instead (12). Anti-OspA in combination with anti-Flagellin may be an exemplary choice in the analysis of either nucleic acid data or IFA, as these two proteins constitute one-third of the total protein content of a spirochete during early Lyme disease (54, 55). The gene expression profile of long-term persisters within a host is as yet unknown.
Recently, another study was published in which Borrelia spirochetes appeared to be present in the form of biofilms in human brain specimens of a chronic Lyme disease case. This study refers to the usage of a monoclonal antibody that is specific for B. burgdorferi sensu stricto (56), yet there was no reference to a commercial source or a research laboratory. The methodology section of the paper cites articles that used a conjugated version of rabbit-polyclonal antibodies which target Borrelia spirochetes. The study neglected to include controls testing cross-reactivity of the antibodies used, so it is difficult to determine the validity of the IFA and to repeat the assay. The authors, however, indicated that Borrelia sequences were identified from the tissues through metagenomics sequencing.
In the study reported here, we used primers that target internal transcribed spacer region (ITS) of the bacterial ribosomal RNA. Although the protein coding regions often have a higher specificity compared to ribosomal markers (57), low PCR amplification, integrity of the tissue sample, and low copy number eliminated them as candidates for the PCR assay of our human autopsy specimens. Previously, 16S rRNA gene was utilized for rapid detection and identification of Borrelia species considering its ubiquity among all the members of the Borrelial genus and almost all bacteria (58). However, this 16S rRNA gene would be very difficult to differentiate between species of Borrelia because of its high sequence similarity. To differentiate Borrelia burgdorferi from other species, we utilized nested PCR. According to a BLAST search, these primers matched 100% with different isolates of B. burgdorferiand didn’t align with any other bacteria or host species except, the Borrelia species finlandensis. According to a recent study in which 7,292 clinical specimens were tested for Borrelia species in US patients, five different species of Borrelia were identified and the species finlandensis was not one of them (59). Most recently, a group that analyzed the microbiomes of ticks collected from the states of New York and Connecticut identified only two Borrelia species, B. burgdorferi and B. miyamotoi, in adult Ixodes scapularis ticks (60). Out of 197 ticks that were analyzed, B. burgdorferi was detected in 111 (56.3%) of the individual ticks and B. miyamotoi in 10 (5.07%) ticks. Among these 10 ticks, seven ticks harbored both species (60). Considering the geographical location and the environment of the Lyme case used in this study and the tick microbiome study, designing primers that are sensitive and specifically detect B. burgdorferi was of utmost importance.
Given the disparity in findings over multiple studies, having multiple methodologies to evaluate specimens for Bb should significantly strengthen any results. Studies suggesting a role for Bb in dementia have been published previously by (38, 46, 47, 61, 62), but negative findings for Borrelia spirochetes have also been reported by others as mentioned above (43, 63). Our studies here represent a major improvement in methodology– both in terms of microbial probing techniques and in numbers of brain samples.
In this report, we provide methodology which succeeded in identifying persistent Borrelia in the CNS of a deceased woman with a history of Lyme disease. This patient did not meet full diagnostic criteria for neuroborreliosis, as it was never demonstrated that she had B. burgdorferi– specific intrathecal antibody production, nor did her CSF show lymphocytosis. While she did have 4 IgG Bb-specific IgG bands in her CSF when assessed by Western blot, specific intrathecal production which requires a comparison of serum and CSF by a diagnostic ELISA was never assessed. The lack of CSF lymphocytosis may reflect the prior extensive antibiotic therapy. Our molecular results however confirm B. burgdorferi invasion of the central nervous system. An earlier lumbar puncture at the time of the initial cognitive decline and prior to the intravenous antibiotic therapy may have confirmed the diagnosis of neuroborreliosis; this case highlights the clinical importance of CSF studies before initiating antibiotic therapy for presumed neurologic Lyme disease. Her initial good response to the IV ceftriaxone suggests a microbial infection was being treated, or that inflammation was dampened. The decline thereafter suggests either that persister Borrelia were present that are now known not to remit with standard antibiotic therapy (6, 12), that an irreversible neurodegenerative process had been triggered by the prior B. burgdorferi infection, or that an unrelated neurodegenerative disorder was present at the same time as the presumed B. burgdorferi CNS infection.
A prior case series of patients who developed chronic neurologic Lyme disease in the United States (64) noted that encephalopathy may emerge months to many years after treated erythema migrans and that about 22% of these patients with late neurologic manifestations show an initial improvement in cognition after intravenous ceftriaxone therapy that is followed months later by relapse. Our patient demonstrated severe headache at the time of the EM rash which suggests meningeal inflammation, a symptom profile also reported by 41% of the patients at initial infection in the case series of patients who later developed chronic neurologic Lyme disease. Notably, our patient did have a good response to the antibiotic treatment only to develop a sleep disorder 2 years later and a cognitive disorder 4 years later.
This patient’s neurodegenerative disorder demonstrated clinical (REM behavior disorder, visuospatial, and attention problems) and neuropathologic features of a Lewy Body Dementia. The case report raises the question of whether B. burgdorferi may play a role in the development of Lewy body dementia. Future studies will be directed at testing more affected subjects and more control subjects in order to substantiate or refute this possible link.