The debate between post-treatment Lyme disease syndrome (PTLDS) and chronic Lyme disease (CLD) reflects different views about the causes and treatment of persistent symptoms attributed to Lyme disease, including symptoms that continue after recommended antibiotic treatment. This study examines how competing positions in the controversy draw on different scientific evidence domains, and how findings from those domains are extended into broader clinical and mechanistic claims. We analysed a 2000–2024 literature corpus using a prompt-optimised three-model large language model ensemble to classify abstracts by stance, theme and model-derived study design. We then conducted targeted full-text audits of selected retreatment and treatment-duration trials, and preclinical persistence studies. Across the higher-volume evidence domains, model-derived claim orientations differed by study-design tier. Observational studies and commentaries showed PTLDS-oriented distributions, whereas case reports or series, animal models, and in vitro studies showed CLD-oriented distributions. The smaller RCT and guideline tiers were also PTLDS-oriented, but the small numbers and wide confidence intervals made this direction uncertain. The clinical trial audit showed that some trials reported short-term or symptom-specific improvements, while the interpretation of these findings depended on their durability, endpoint consistency, eligibility criteria, treatment burden, and safety. The citation-conditioned preclinical audit identified findings that support several candidate mechanisms and generate hypotheses for further study, but these findings did not by themselves establish viable infection as the cause of persistent human symptoms or demonstrate the efficacy of prolonged antimicrobial treatment. The findings show a recurring distinction between mechanism-level evidence and the evidence needed to establish patient-level causation or durable clinical benefit. They map how these different forms of evidence are distributed and translated across the PTLDS–CLD literature.

Lyme borreliosis (LB), commonly referred to as Lyme disease (LD), is a prominent global health issue, exhibiting a seroprevalence rate of 14.5%. Heightened incidence levels of LD have been recorded in parts of Europe, Poland, Eastern Europe, and the Baltic States. The research aimed to inform the cost of LD and post-treatment Lyme disease syndrome (PTLDS) in Ireland through results from a patient questionnaire, disease modelling, the construction of a patient roadmap, and attempts to arrive at prevalence calculation estimates based on local data. Patient data encompassed sociodemographic particulars, disease attributes, healthcare resource utilization, and the influence on their employment status. Of 301 patients, 210 were diagnosed with LD and/or a tick-borne infection (TBI), the cohort’s average age was 40.07 (SD 13.5) (N = 210; Female:Male 60:40). The mean duration of symptoms in PTLDS patients was 7.15 years. The average number of visits to other healthcare professionals was 16.8 per patient. Regarding current employment status, the data indicates that 50.2% of respondents were currently working, 10.1% were unemployed, 8.7% were retired, 5.3% had caring responsibilities, 11.1% were on sick leave, and 14.5% fell into the “Other” category. Additionally, when asked if symptoms had affected their employment status, 69% of respondents said yes, 26% said no, and 5% did not respond. Modeling efforts show that the roadmap to care for PTLDS is challenging, leading to wandering from specialty to specialty and high healthcare utilization. Utilizing a novel method of indirect reverse estimation, our lifetime risk or cumulative incidence of PTLDS estimation is at 0.003%. Lack of data collection from Irish health authorities is leaving the issue of the cost of LD and PTLDS hard to address, despite efforts from our single-site study.

Background/Objectives:Lyme disease (LD) and its co-infections present significant diagnostic and treatment challenges due to their complex interplay with neurological symptoms, immune responses, autoimmune reactions, and mental health conditions. Standard two-tier LD testing often fails to detect cases, necessitating expanded serologic and functional testing. Following CARE Guidelines, this case series examines ten clinical narratives of LD, highlighting the limitations of standard diagnostic methods, the potential benefits of specialized testing, and the need for an effective paradigm for LD management. Methods:A multidisciplinary approach grounded in integrative medicine was adopted. Diagnostic methods included advanced serological panels (e.g., TICKPLEX®), co-infection testing, functional immune markers, Cunningham Panel testing for neuropsychiatric Lyme presentations, and imaging. Treatments included traditional antibiotics, botanical antimicrobials (Cryptolepis, Artemisinin, Biocidin), mitochondrial and neuroprotective support (CoQ10, NAD+, magnesium), nutritional therapies, detox support, immune system support protocols, pulsed electromagnetic field therapy, and methylene blue protocols. Results:The findings from this case series demonstrate the heterogeneity of LD manifestations across different ages, genders, and backgrounds. Compared to promising specialized testing, standard laboratory tests often misdiagnose LD and its co-infections as Parkinson's disease, multiple sclerosis, lupus, autism, and psychiatric disorders. Furthermore, integrating diverse treatment modalities, including combination and rotational antibiotic therapy, IV ceftriaxone for neurological cases, detoxification support (glutathione IV, vitamin C IV), and immune modulation with intravenous immunoglobulin, helped manage symptoms. Herxheimer reactions were effectively managed with antioxidant and detox therapies. Patients receiving long-term maintenance therapies, including herbal antimicrobials and gut microbiome support, showed fewer relapses. Conclusions:This case series advocates for a holistic, patient-centered approach. It emphasizes the necessity of comprehensive diagnostics that consider external factors, including post-vaccine symptom exacerbations, immune dysregulation, personalized treatment strategies, and ongoing research to improve LD management. These findings provide an evidence-based framework for physicians to integrate conventional and natural medicine strategies to optimize LD care.

Tick bites acquired in the northern or southern hemisphere can transmit microbes that may cause illness. The most prevalent infection is Lyme borreliosis (LB), with all proven cases to date having been acquired in the northern hemisphere. The existence of endemic LB in Australia has not been proven explicitly, and there is uncertainty concerning the cause of “Lyme-like” disease (LLD) in Australia. As many tick-borne diseases (TBDs) are diagnosed by serology, validated assays for use in both the northern and southern hemispheres are required. Using a multiplex enzyme-linked immunosorbent assay (TICKPLEX®), two independent laboratories tested a total of 53 well-characterized reference sera that consisted of 33 samples from northern hemisphere patients with confirmed tick-borne disease (TBD) and 20 randomly selected sera from Australian patients with suspected TBDs, presenting with or without LLD. Antibody responses to multiple microbial antigens from causative agents of TBDs were found. High concordance between laboratories was demonstrated on this small set of samples. The results obtained provide the basis for further evaluation of TICKPLEX® on a larger number of samples from Australian patients with suspected TBDs. These findings should be considered preliminary, providing proof-of-concept evidence that warrants validation in larger, clinically diverse cohorts.

The scientific discourse surrounding Chronic Lyme Disease (CLD) and Post-Treatment Lyme Disease Syndrome (PTLDS) has evolved over the past twenty-five years into a complex and polarised debate, shaped by shifting research priorities, institutional influences, and competing explanatory models. This study presents the first large-scale, systematic examination of this discourse using an innovative hybrid AI-driven methodology, combining large language models with structured human validation to analyse thousands of scholarly abstracts spanning 25 years. By integrating Large Language Models (LLMs) with expert oversight, we developed a quantitative framework for tracking epistemic shifts in contested medical fields, with applications to other content analysis domains. Our analysis revealed a progressive transition from infection-based models of Lyme disease to immune-mediated explanations for persistent symptoms. This study offers new empirical insights into the structural and epistemic forces shaping Lyme disease research, providing a scalable and replicable methodology for analysing discourse, while underscoring the value of AI-assisted methodologies in social science and medical research.

Tick bites acquired in the northern or southern hemispheres can transmit microbes that may cause illness. The most prevalent infection is Lyme Disease, with all proven cases to date having been acquired in the northern hemisphere. The existence of endemic Lyme disease (LD) in Australia has not been proven explicitly, and there is uncertainty concerning the cause of “Lyme-like” disease (LLD) in Australia. As many tick-borne infections (TBIs) are diagnosed by serology, validated assays for use in both the northern and southern hemispheres are required. Using a multiplex enzyme-linked immunosorbent assay (TICKPLEX®), two independent laboratories tested a total of 53 well-characterized reference sera that consisted of 33 samples from northern hemisphere patients with confirmed tick-borne disease (TBD) and 20 randomly selected sera from Australian patients with suspected TBIs, presenting with or without LLD. Antibody responses to multiple microbial antigens from causative agents of TBDs were found. High concordance between laboratories was demonstrated on this small set of samples. The results obtained provide the basis for further evaluation of TICKPLEX® on a larger number of samples from Australian patients with suspected TBIs.

The field of data analysis, preparation, and machine learning is rapidly expanding, offering numerous libraries and resources for exploration. Researchers gain knowledge through various channels, but few resources provide a comprehensive framework for building machine-learning models. We present a step-by-step framework for constructing a robust Random Forest classification model to fill this gap. Using the trained model, we predict if individuals visiting Sanoviv Medical Institute between 2020 and 2023 participated in the Lyme disease program based on age, symptoms, blood count, and chemistry results. While not exhaustive, the methods in each step provide a valuable starting point for researchers, promoting an understanding of the fundamental approach to model creation. The framework encourages researchers to explore beyond the outlined techniques, fostering innovation and experimentation.

Tick-borne illnesses (TBIs), especially those caused by Borrelia, are increasingly prevalent worldwide. These diseases progress through stages of initial localization, early spread, and late dissemination. The final stage often leads to post-treatment Lyme disease syndrome (PTLDS) or chronic Lyme disease (CLD), characterized by persistent and non-specific multisystem symptoms affecting multiple systems, lasting over six months after antibiotic therapy. PTLDS significantly reduces functional ability, with 82-96% of patients experiencing pain, including arthritis, arthralgia, and myalgia. Inflammatory markers like CRP and TNF-alpha indicate ongoing inflammation, but the link between chronic pain and other biomarkers is underexplored. This study examined the relationship between pain and biomarkers in TBI patients from an Irish hospital and their response to antibiotic treatment. Pain ratings significantly decreased after antibiotic treatment, with median pain scores dropping from 7 to 5 (U = 27215.50, p < 0.001). This suggests a persistent infection responsive to antibiotics. Age and gender did not influence pain ratings before and after treatment. The study found correlations between pain ratings and biomarkers such as transferrin, CD4%, platelets, and neutrophils. However, variations in these biomarkers did not significantly predict pain changes when considering biomarkers outside the study. These findings imply that included biomarkers do not directly predict pain changes, possibly indicating allostatic load in symptom variability among long-term TBI patients. The study emphasizes the need for appropriate antibiotic treatment for TBIs, highlighting human rights issues related to withholding pain relief.

Standard clinical markers can improve tick-borne infection (TBI) diagnoses. We investigated immune and other clinical biomarkers in 110 patients clinically diagnosed with TBIs before (T0) and after antibiotic treatment (T2). At T0, both the initial observation group and patients without seroconversion for tick-borne pathogens exhibited notably low percentages and counts of CD3 percentage (CD3%), CD3+ cells, CD8+ suppressors, CD4 percentage (CD4%), and CD4+ helper cells, with the latter group showing reductions in CD3%, CD3+, and CD8+ counts in approximately 15-22% of cases. Following treatment at the T2 follow-up, patients typically experienced enhancements in their previously low CD3%, CD3+ counts, CD4%, and CD4+ counts; however, there was no notable progress in their low CD8+ counts, and a higher number of patients presented with insufficient transferrin levels. Moreover, among those with negative serology for tick-borne infections, there was an improvement in low CD3% and CD3+ counts, which was more pronounced in patients with deficient transferrin amounts. Among those with CD57+ (n = 37) and CD19+ (n = 101) lymphocyte analysis, 59.46% of patients had a low CD57+ count, 14.85% had a low CD19 count, and 36.63% had a low CD19 percentage (CD19%). Similar findings were observed concerning low CD57+, CD19+, and CD19% markers for negative TBI serology patients. Overall, this study demonstrates that routine standard clinical markers could assist in a TBI diagnosis.

Emerging organoid research is paving way for studies in infectious diseases. Described here is a technique for the generation of stem-cell derived organoids for human small intestine and lung together with methods to infect such organoids with a mock pathogen (Cryptosporidium parvum). Such systems are amenable to imaging and processing for molecular biological analyses. It is the intent of this chapter to provide a simple, routine organoid procedure so that in vitro studies with Borrelia such as cell invasion and dissemination can be conducted.

The rising prevalence of tick-borne infections (TBIs) necessitates further attention. This study retrospectively investigated the types of TBIs, symptoms, and if combination antibiotics were helpful within a patient cohort at an infectious disease clinic in Ireland. In this chart audit of 301 individuals (184 female, 117 male) tested for TBIs, 140 (46.51%) had positive antibody responses for TBIs from an ELISA (enzyme-linked immunoassay) that was based on a modified two-tiered testing protocol. A total of 93 (66.43%) patients had positive antibody responses to one TBI: 83 (59.29%) for Borrelia, 7 (5.00%) for Rickettsia, and 1 (0.71%) each for either Babesia, Bartonella, or Ehrlichia. The remaining 47 (33.57%) patients were infected with multiple TBIs. These patients were treated with combination antibiotics and monitored at two subsequent follow-ups. Only 2 of 101 patients (1.98%) had discontinued treatment by the second follow-up. In the first follow-up with 118 patients, 70 (59.32%) reported pain and 48 (40.68%) had neurological symptoms. In the next follow-up of 101 patients, 41 (40.59%) had pain while 30 (29.70%) had neurological symptoms. There were statistically significant reductions in the incidence of pain (41.43%) and neurological (37.50%) symptoms between follow-ups. Thus, our study demonstrates that combination antibiotics effectively relieve TBI symptoms with good patient tolerance.

Currently, there are over 602 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and 6.4 million COVID-19 disease-related deaths worldwide. With ambitious vaccine strategies, reliable and accurate serological testing is needed to monitor the dynamics of the novel coronavirus pandemic and community immunity. We set out to improve serological testing of the immune response against SARS-CoV-2. We hypothesize that by multiplexing the serological diagnostic test kit (SARSPLEX) and screening for three antibodies, an even more robust diagnostic can be developed. A total of 293 sera were analyzed for IgM, IgG, or IgA immune reactions to the subunit 1 spike glycoprotein and the nucleocapsid protein in a standardized ELISA platform. Testing IgM, IgG, and IgA demonstrated high positive and negative agreements compared to RT-PCR and serology reference tests. Comparison with the pre-2019-CoV (n = 102) samples highlighted the specificity of this test kit and indicated that no unspecific binding, even with the summer flu patients (n = 44), was detected. In addition, SARSPLEX demonstrated to be a valuable occupational surveillance tool used in a functional medicine facility. With increased and broader testing, SARSPLEX will be a valuable tool in monitoring immunity and aid in prioritizing access to the SARS-CoV-2 vaccine for high-risk patients.

Mycotoxin exposure in humans is primarily assessed through its occurrence in external sources, such as food commodities. Herein, we have developed a direct competitive ELISA to facilitate the detection of aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin (FUM B1/B2), ochratoxin A (OTA), and zearalenone (ZEA) in human serum. The analytical validation of the assay followed practices endorsed by the international research community and the EU directive 96/23/EC in order to examine detection capability, recovery, and cross-reactivity. The assay demonstrated a lower limit of quantitation (LLOQ) for AFB1 [0.61 ng/mL (hereon ng/mL = ppb)], DON (19.53 ppb), FUM (4.88 ppb), OTA (19.53 ppb), and ZEA (0.15 ppb). Recovery from human serum for all mycotoxins spanned from 73% to 106%. Likewise, the specificity for monoclonal antibodies against cross-reactant mycotoxins ranged from 2% to 11%. This study compares the LLOQ and recovery values with commercial and emerging immuno-based methods for detecting mycotoxins in foodstuffs. The LLOQ values from the present study were among the lowest in commercial or emerging methods. Despite the differences in the extraction protocols and matrices, the recovery range in this study, commercial tests, and other procedures were similar for all mycotoxins. Overall, the assay detected AFB1, DON, FUM, OTA, and ZEA in human serum with excellent accuracy, precision, and specificity.

Human polymicrobial infections in tick-borne disease (TBD) patients is an emerging public health theme. However, the requirement for holistic TBD tests in routine clinical laboratories is ambiguous. TICKPLEX® PLUS is a holistic TBD test utilized herein to assess the need for multiplex and multifunctional diagnostic tools in a routine clinical laboratory. The study involved 150 specimens categorized into Lyme disease (LD)-positive (n = 48), LD-negative (n = 30), and febrile patients from whom borrelia serology was requested (n = 72, later "febrile patients") based on reference test results from United Medix, Finland. Reference tests from DiaSorin, Immunetics, and Mikrogen Diagnostik followed the two-tier LD testing system. A comparison between the reference tests and TICKPLEX® PLUS produced 86%, 88%, and 87% positive, negative, and overall agreement, respectively. Additionally, up to 15% of LD and 11% of febrile patients responded to TBD related coinfections and opportunistic microbes. The results demonstrated that one (TICKPLEX® PLUS) test can aid in a LD diagnosis instead of four tests. Moreover, TBD is not limited to just LD, as the specimens produced immune responses to several TBD microbes. Lastly, the study indicated that the screening of febrile patients for TBDs could be a missed opportunity at reducing unreported patient cases.

Abstract Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos‐methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance‐associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos‐methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.

Lyme borreliosis is a multisystemic disease caused by the pleomorphic bacteria of the Borrelia burgdorferi sensu lato complex. The exact mechanisms for the infection to progress into a prolonged sequelae of the disease are currently unknown, although immune evasion and persistence of the bacteria in the host are thought to be major contributors. The current study investigated B. burgdorferi infection processes in two human cell lines, both non-immune and non-phagocytic, to further understand the mechanisms of infection of this bacterium. By utilizing light, confocal, helium ion, and transmission electron microscopy, borrelial infection of chondrosarcoma (SW1353) and dermal fibroblast (BJ) cells were examined from an early 30-min time point to a late 9-days post-infection. Host cell invasion, viability of both the host and B. burgdorferi, as well as, co-localization with lysosomes and the presence of different borrelial pleomorphic forms were analyzed. The results demonstrated differences of infection between the cell lines starting from early entry as B. burgdorferi invaded BJ cells in coiled forms with less pronounced host cell extensions, whereas in SW1353 cells, micropodial interactions with spirochetes were always seen. Moreover, infection of BJ cells increased in a dose dependent manner throughout the examined 9 days, while the percentage of infection, although dose dependent, decreased in SW1353 cells after reaching a peak at 48 h. Furthermore, blebs, round body and damaged B. burgdorferi forms, were mostly observed from the infected SW1353 cells, while spirochetes dominated in BJ cells. Both infected host cell lines grew and remained viable after 9 day post-infection. Although damaged forms were noticed in both cell lines, co-localization with lysosomes was low in both cell lines, especially in BJ cells. The invasion of non-phagocytic cells and the lack of cytopathic effects onto the host cells by B. burgdorferi indicated one mechanism of immune evasion for the bacteria. The differences in attachment, pleomorphic form expressions, and the lack of lysosomal involvement between the infected host cells likely explain the ability of a bacterium to adapt to different environments, as well as, a strategy for persistence inside a host.

The present study evaluates the role of graphene oxide’s (GO’s) peroxidase-like and inherent/carbocatalytic properties in oxidising silver nitrate (AgNO 3 ) to create graphene nanocomposites with silver nanoparticles (GO/Ag nanocomposite). Activation of peroxidase-like catalytic function of GO required hydrogen peroxide (H 2 O 2 ) and ammonia (NH 3 ) in pH 4.0 disodium hydrogen phosphate (Na 2 HPO 4 ). Carbocatalytic abilities of GO were triggered in pH 4.0 deionised distilled water (ddH 2 O). Transmission electron microscope (TEM), scanning electron microscope (SEM), cyclic voltammetry (CV) and UV-Vis spectroscopy aided in qualitatively and quantitatively assessing GO/Ag nanocomposites. TEM and SEM analysis demonstrated the successful use of GO’s peroxidase-like and carbocatalytic properties to produce GO/Ag nanocomposite. UV-Vis analysis indicated a higher yield in optical density values for GO/Ag nanocomposites created using GO’s carbocatalytic ability rather than its peroxidase-like counterpart. Additionally, CV demonstrated that GO/Ag nanocomposite fabricated here is a product of an irreversible electrochemical reaction. Our study outcomes show new opportunities for GO as a standalone catalyst in biosensing. We demonstrate a sustainable approach to obtain graphene nanocomposites exclusive of harmful chemicals or physical methods.

Introduction: The tick-borne disease Lyme Borreliosis is caused by Borrelia bacteria. The disease can persist even after treatment with antibiotics, which is why other methods of treatment are needed. Herbal compounds and phytochemicals have been recently examined in relation to eradicating Borrelia bacteria in vitro. Objective: The possible antimicrobial effect of two novel compounds, Biocidin Liquid and LSF Broad-Spectrum Liposomal formulas, was examined in the hopes of discovering an alternative method for eradication of Borrelia bacteria. Methods: The minimum inhibitory concentrations (MICs) and minimum bacterial deaths (MBDs), as well as, time-kill effect of each compound were utilized in the study. Results: The Liquid formula effectively killed the spirochetes with 1:10 dilution, while the MIC for the Liposomal formula was 1:25. Moreover, the MIC for both compounds with Round Bodies was 1:50 and for biofilms 1:10. Though long-term effect (MBD) was seen only with 1:5 dilutions for both formulas. Additionally, the killing effect of each compound was observed already at 10 min post-treatment. Conclusion: The study conducted here provides new insight into the antimicrobial effect of herbal compounds. Furthermore, studies such as these are required in order to discover possible alternatives to antibiotics in the battle against Borrelia infections. *Correspondence to: Kati Karvonen, M.Sc, Department of Biological and Environmental Science and Nanoscience Center, PO Box 35, FI-40014, University of Jyvaskyla, Finland, E-mail: kati.s.karvonen@jyu.fi

BACKGROUND:Viral infection is implicated in development of autoimmunity. Parvovirus B19 (B19V) nonstructural protein, NS1, a helicase, covalently modifies self double-stranded deoxyribonucleic acid (dsDNA) and induces apoptosis. This study tested whether resulting apoptotic bodies (ApoBods) containing virally modified dsDNA could induce autoimmunity in an animal model. METHODS:BALB/c mice were inoculated with (1) pristane-induced, (2) B19V NS1-induced, or (3) staurosporine-induced ApoBods. Serum was tested for dsDNA autoantibodies by Crithidia luciliae staining and enzyme-linked immunosorbent assay. Brain, heart, liver, and kidney pathology was examined. Deposition of self-antigens in glomeruli was examined by staining with antibodies to dsDNA, histones H1 and H4, and TATA-binding protein. RESULTS:The B19V NS1-induced ApoBod inoculation induced dsDNA autoantibodies in a dose-dependent fashion. Histopathological features of immune-mediated organ damage were evident in pristane-induced and NS1-induced ApoBod groups; severity scores were higher in these groups than in staurosporine-treated groups. Tissue damage was dependent on NS1-induced ApoBod dose. Nucleosomal antigens were deposited in target tissue from pristane-induced and NS1-induced ApoBod inoculated groups, but not in the staurosporine-induced ApoBod inoculated group. CONCLUSIONS:This study demonstrated proof of principle in an animal model that virally modified dsDNA in apoptotic bodies could break tolerance to self dsDNA and induce dsDNA autoantibodies and end-organ damage.