Dried Blood Microsampling-Based Therapeutic Drug Monitoring of Antiepileptic Drugs in Children With Nodding Syndrome and Epilepsy in Uganda and the Democratic Republic of the Congo.

Nodding syndrome is a highly debilitating, generalized seizure disorder, affecting children in subregions of sub-Saharan Africa. Despite numerous efforts to uncover the etiology, the exact cause of this syndrome still remains obscure. Therefore, to date, patients only receive symptomatic care, including the administration of first-generation antiepileptic drugs for seizure control. As data on the efficacy of drugs within this population are completely lacking, the aim of this study was to explore how therapeutic drug monitoring could help to understand the differential response to therapy. Considering the challenging environment in which sampling had to be performed (remote areas, devoid of electricity, running water, etc), dried blood matrices [ie, dried blood spots (DBSs)] and volumetric absorptive microsampling (VAMS) were considered fit-for-purpose. In addition, owing to the similarities between the syndrome and other forms of epilepsy, samples originating from patients suffering from (onchocerciasis-associated) epilepsy were included. In total, 68 patients with Nodding syndrome from Uganda, 58 Ugandan patients with epilepsy, and 137 patients with onchocerciasis-associated epilepsy from the Democratic Republic of the Congo were included. VAMS samples and DBS were analyzed using validated methods, involving manual extraction or fully automated extraction, respectively, before quantification using liquid chromatography coupled with tandem mass spectrometry. Analysis revealed that serum concentrations (calculated from DBS) within the respective reference ranges were attained in only 52.9% of the 68 Nodding syndrome patients treated with valproic acid, in 21.4% of the 56 Ugandan epilepsy patients treated with carbamazepine, and in 65.7% of the 137 onchocerciasis-associated epilepsy patients from the Democratic Republic of the Congo treated with phenobarbital. In all other instances, concentrations were subtherapeutic. Furthermore, on comparing DBS with VAMS concentrations, an inexplicable overestimation was observed in the latter. Finally, no obvious link could be observed between the obtained drug concentrations and the number of seizures experienced during the last month before sampling, elaborating the fact that the level of improvement in some patients cannot simply be linked to reaching therapeutic concentrations.

Colistin-Resistant Acinetobacter baumannii Clinical Strains with Deficient Biofilm Formation.

In two pairs of clinical colistin-susceptible/colistin-resistant (Cst(s)/Cst(r)) Acinetobacter baumannii strains, the Cst(r) strains showed significantly decreased biofilm formation in static and dynamic assays (P < 0.001) and lower relative fitness (P < 0.05) compared with those of the Cst(s) counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cst(r) strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other.

The natural history of CVB3 myocarditis in C57BL/6J mice: an extended in-depth characterization.

BACKGROUND AND AIMS: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course. METHODS AND RESULTS: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 105 (regular dose) (RD) or 5 × 106 (high dose) (HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8, and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<.001). In male CVB mice, premature mortality occurred between days 8 and 23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups. CONCLUSION: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease.

Host immunological responses facilitate development of SARS-CoV-2 mutations in patients receiving monoclonal antibody treatments.

BackgroundThe role of host immunity in emergence of evasive SARS-CoV-2 Spike mutations under therapeutic monoclonal antibody (mAb) pressure remains to be explored.MethodsIn a prospective, observational, monocentric ORCHESTRA cohort study, conducted between March 2021 and November 2022, mild-to-moderately ill COVID-19 patients (n = 204) receiving bamlanivimab, bamlanivimab/etesevimab, casirivimab/imdevimab, or sotrovimab were longitudinally studied over 28 days for viral loads, de novo Spike mutations, mAb kinetics, seroneutralization against infecting variants of concern, and T cell immunity. Additionally, a machine learning-based circulating immune-related biomarker (CIB) profile predictive of evasive Spike mutations was constructed and confirmed in an independent data set (n = 19) that included patients receiving sotrovimab or tixagevimab/cilgavimab.ResultsPatients treated with various mAbs developed evasive Spike mutations with remarkable speed and high specificity to the targeted mAb-binding sites. Immunocompromised patients receiving mAb therapy not only continued to display significantly higher viral loads, but also showed higher likelihood of developing de novo Spike mutations. Development of escape mutants also strongly correlated with neutralizing capacity of the therapeutic mAbs and T cell immunity, suggesting immune pressure as an important driver of escape mutations. Lastly, we showed that an antiinflammatory and healing-promoting host milieu facilitates Spike mutations, where 4 CIBs identified patients at high risk of developing escape mutations against therapeutic mAbs with high accuracy.ConclusionsOur data demonstrate that host-driven immune and nonimmune responses are essential for development of mutant SARS-CoV-2. These data also support point-of-care decision making in reducing the risk of mAb treatment failure and improving mitigation strategies for possible dissemination of escape SARS-CoV-2 mutants.FundingThe ORCHESTRA project/European Union's Horizon 2020 research and innovation program.

Assessing Onchocerca volvulus Intensity of Infection and Genetic Diversity Using Mitochondrial Genome Sequencing of Single Microfilariae Obtained before and after Ivermectin Treatment.

Onchocerciasis is a neglected tropical disease targeted for elimination using ivermectin mass administration. Ivermectin kills the microfilariae and temporarily arrests microfilariae production by the macrofilariae. We genotyped 436 microfilariae from 10 people each in Ituri, Democratic Republic of the Congo (DRC), and Maridi County, South Sudan, collected before and 4-5 months after ivermectin treatment. Population genetic analyses identified 52 and 103 mitochondrial DNA haplotypes among the microfilariae from DRC and South Sudan, respectively, with few haplotypes shared between people. The percentage of genotype-based correct assignment to person within DRC was ~88% and within South Sudan ~64%. Rarefaction and extrapolation analysis showed that the genetic diversity in DRC, and even more so in South Sudan, was captured incompletely. The results indicate that the per-person adult worm burden is likely higher in South Sudan than DRC. Analyses of haplotype data from a subsample (n = 4) did not discriminate genetically between pre- and post-treatment microfilariae, confirming that post-treatment microfilariae are not the result of new infections. With appropriate sampling, mitochondrial haplotype analysis could help monitor changes in the number of macrofilariae in a population as a result of treatment, identify cases of potential treatment failure, and detect new infections as an indicator of continuing transmission.

Procedure for Handling and Storage of Onchocerca volvulus Microfilariae Obtained from Skin Snips for Downstream Genetic Work.

WHO and endemic countries target elimination of transmission of Onchocerca volvulus, the parasite causing onchocerciasis. Population genetic analysis of O. volvulus may provide data to improve the evidence base for decisions on when, where, and for how long to deploy which interventions and post-intervention surveillance to achieve elimination. Development of necessary methods and tools requires parasites suitable for genetic analysis. Based on our experience with microfilariae obtained from different collaborators, we developed a microfilariae transfer procedure for large-scale studies in the Democratic Republic of Congo (DRC) comparing safety and efficacy of ivermectin, the mainstay of current onchocerciasis elimination strategies, and moxidectin, a new drug. This procedure is designed to increase the percentage of microfilariae in skin snips suitable for genetic analysis, improve assignment to metadata, and minimize time and materials needed by the researchers collecting the microfilariae. Among 664 microfilariae from South Sudan, 35.7% and 39.5% failed the mitochondrial and nuclear qPCR assay. Among the 576 microfilariae from DRC, 16.0% and 16.7% failed these assays, respectively. This difference may not only be related to the microfilariae transfer procedure but also to other factors, notably the ethanol concentration in the tubes in which microfilariae were stored (64% vs. ≥75%).

Immunogenicity and reactogenicity of a first booster with BNT162b2 or full-dose mRNA-1273: A randomised VACCELERATE trial in adults ≥75 years (EU-COVAT-1).

BACKGROUND: Vaccination remains crucial for protection against severe SARS-CoV-2 infection, especially for people of advanced age, however, optimal dosing regimens are as yet lacking. METHODS: EU-COVAT-1-AGED Part A is a randomised controlled, adaptive, multicentre phase II trial evaluating safety and immunogenicity of a 3rd vaccination (1st booster) in individuals ≥75 years. Fifty-three participants were randomised to full-doses of either mRNA-1273 (Spikevax®, 100 µg) or BNT162b2 (Comirnaty®, 30 µg). The primary endpoint was the rate of 2-fold circulating antibody titre increase 14 days post-vaccination measured by quantitative electrochemiluminescence (ECL) immunoassay, targeting RBD region of Wuhan wild-type SARS-CoV-2. Secondary endpoints included the changes in neutralising capacity against wild-type and 25 variants of concern at 14 days and up to 12 months. Safety was assessed by monitoring of solicited adverse events (AEs) for seven days after on-study vaccination. Unsolicited AEs were collected until the end of follow-up at 12 months, SAEs were pursued for a further 30 days. RESULTS: Between 08th of November 2021 and 04th of January 2022, 53 participants ≥75 years received a COVID-19 vaccine as 1st booster. Fifty subjects (BNT162b2 n = 25/mRNA-1273 n = 25) were included in the analyses for immunogenicity at day 14. The primary endpoint of a 2-fold anti-RBD IgG titre increase 14 days after vaccination was reached for all subjects. A 3rd vaccination of full-dose mRNA-1273 provided higher anti-RBD IgG titres (Geometric mean titre) D14 mRNA-127310711 IU/mL (95 %-CI: 8003;14336) vs. BNT162b2: 7090 IU/mL (95 %-CI: 5688;8837). We detected a pattern showing higher neutralising capacity of full-dose mRNA-1273 against wild-type as well as for 23 out of 25 tested variants. INTERPRETATION: Third doses of either BNT162b2 or mRNA-1273 provide substantial circulating antibody increase 14 days after vaccination. Full-dose mRNA-1273 provides higher antibody levels with an overall similar safety profile for people ≥75 years. FUNDING: This trial was funded by the European Commission (Framework Program HORIZON 2020).

Using machine learning to predict antibody response to SARS-CoV-2 vaccination in solid organ transplant recipients: the multicentre ORCHESTRA cohort.

OBJECTIVES: The study aim was to assess predictors of negative antibody response (AbR) in solid organ transplant (SOT) recipients after the first booster of SARS-CoV-2 vaccination. METHODS: Solid organ transplant recipients receiving SARS-CoV-2 vaccination were prospectively enrolled (March 2021-January 2022) at six hospitals in Italy and Spain. AbR was assessed at first dose (t0), second dose (t1), 3 ± 1 month (t2), and 1 month after third dose (t3). Negative AbR at t3 was defined as an anti-receptor binding domain titre <45 BAU/mL. Machine learning models were developed to predict the individual risk of negative (vs. positive) AbR using age, type of transplant, time between transplant and vaccination, immunosuppressive drugs, type of vaccine, and graft function as covariates, subsequently assessed using a validation cohort. RESULTS: Overall, 1615 SOT recipients (1072 [66.3%] males; mean age±standard deviation [SD], 57.85 ± 13.77) were enrolled, and 1211 received three vaccination doses. Negative AbR rate decreased from 93.66% (886/946) to 21.90% (202/923) from t0 to t3. Univariate analysis showed that older patients (mean age, 60.21 ± 11.51 vs. 58.11 ± 13.08), anti-metabolites (57.9% vs. 35.1%), steroids (52.9% vs. 38.5%), recent transplantation (<3 years) (17.8% vs. 2.3%), and kidney, heart, or lung compared with liver transplantation (25%, 31.8%, 30.4% vs. 5.5%) had a higher likelihood of negative AbR. Machine learning (ML) algorithms showing best prediction performance were logistic regression (precision-recall curve-PRAUC mean 0.37 [95%CI 0.36-0.39]) and k-Nearest Neighbours (PRAUC 0.36 [0.35-0.37]). DISCUSSION: Almost a quarter of SOT recipients showed negative AbR after first booster dosage. Unfortunately, clinical information cannot efficiently predict negative AbR even with ML algorithms.

Immunoinformatics Design and Assessment of a Multiepitope Antigen (OvMCBL02) for Onchocerciasis Diagnosis and Monitoring.

Onchocerciasis is a Neglected Tropical Disease that has a significant socioeconomic impact, especially in Sub-Saharan Africa. Numerous reports indicate that the Expanded Special Project for the Elimination of Neglected Tropical Diseases needs novel diagnostic tools before achieving its goal of successful elimination of onchocerciasis in Africa. The current diagnostic tests are either invasive, insensitive, or not applicable in the field and about 25% of persons infected cannot mount immune responses against the single antigen used in the only approved Ov-16 serological test. In the quest to identify novel biomarkers that can be used to certify that a patient is free from the disease, evaluate the progress of elimination programmes, and conduct post elimination surveillances, mass spectrometric analysis of Onchocerca volvulus crude extract revealed that 1392 proteins are expressed in the adult and microfilariae stages of the parasite. Computational analysis predicted six of the proteins as O. volvulus potential diagnostic targets. Linear B-epitopes were predicted from the six proteins and used to construct a multiepitope antigen (OvMCBL02). Serological analysis revealed that the OvMCBL02 test significantly differentiated between serum samples of onchocerciasis patients from the Kombone Health Area in the South West Region of Cameroon (n = 63) and control serum samples from Rwanda (n = 29) and Europe (n = 26) as well as between serum samples from the onchocerciasis hyperendemic region of Kombone Health Area (n = 63) and the hypoendemic region of Bandjoun Health District (n = 54). Interestingly, the test did not cross-react with serum samples from patients suffering from related nematode infections, thereby suggesting that further characterization of the OvMCBL02 multiepitope antigen will render it an additional member of the diagnostic toolbox for the elimination of onchocerciasis.

Identification of Potential Urinary Metabolite Biomarkers of Pseudomonas aeruginosa Ventilator-Associated Pneumonia.

Introduction: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in hospital intensive care units (ICU). Rapid identification of P. aeruginosa-derived markers in easily accessible patients' samples can enable an early detection of P. aeruginosa VAP (VAP-PA), thereby stewarding antibiotic use and improving clinical outcomes. Methods: Metabolites were analysed using liquid chromatography-mass spectrometry (LC-MS) in prospectively collected urine samples from mechanically ventilated patients admitted to the Antwerp University Hospital ICU. Patients were followed from the start of mechanical ventilation (n = 100 patients) till the time of clinical diagnosis of VAP (n = 13). Patients (n = 8) in whom diagnosis of VAP was further confirmed by culturing respiratory samples and urine samples were studied for semi-quantitative metabolomics. Results: We first show that multivariate analyses highly discriminated VAP-PA from VAP-non-PA as well as from the pre-infection groups (R 2 = .97 and .98, respectively). A further univariate analysis identified 58 metabolites that were significantly elevated or uniquely present in VAP-PA compared to the VAP-non-PA and pre-infection groups (P < .05). These comprised both a known metabolite of histidine as well as a novel nicotine metabolite. Most interestingly, we identified 3 metabolites that were not only highly upregulated for, but were also highly specific to, VAP-PA, as these metabolites were completely absent in all pre-infection timepoints and in VAP-non-PA group. Conclusions: Considerable differences exist between urine metabolites in VAP-PA compared to VAP due to other bacterial aetiologies as well to non-VAP (pre-infection) timepoints. The unique urinary metabolic biomarkers we describe here, if further validated, could serve as highly specific diagnostic biomarkers of VAP-PA.

Host Immunity Influences the Composition of Murine Gut Microbiota.

The influence of gut microbiota on host immunity is widely studied, and its disturbance has been linked to several immune-mediated disorders. Conversely, whether and how inherently disturbed canonical Th1 (pro-inflammatory) and/or Th2 (anti-inflammatory) immune pathways modify the host microbiome is not sufficiently investigated. Here, we characterized the humoral, cellular, and cytokine immunity, and associated alterations in gut microbiota of naïve wild-type mice (C57BL/6 and BALB/c), and mice with deficiencies in Th2 responses (IL-4Rα and IL-33 knockout mice) or in both Th1 and Th2 responses (NOD scid gamma, NSG mice). A global analysis by de novo clustering of 16S rRNA profiles of the gut microbiota independently grouped wild-type immunocompetent (C57BL/6 and BALB/c), Th2-deficient (IL-4Rα-/- and IL-33-/-), and severely immunodeficient (NSG) mice; where wild-type mice, but not Th2 or severely immunodeficient mice, were enriched in gut bacteria that produce short-chain fatty acids. These include members of phyla Firmicutes, Verrucomicrobia, and Bacteroidetes such as Lactobacillus spp., Akkermansia muciniphila, and Odoribacter spp. Further comparison of the two naïve wild-type mouse strains showed higher microbial diversity (Shannon), primarily linked to higher richness (Chao1), as well as a distinct difference in microbial composition (weighted UniFrac) in BALB/c mice compared to C57BL/6. T-cell and blood cytokine analyses demonstrated a Th1-polarization in naïve adaptive immunity in C57BL/6 animals compared to BALB/c mice, and an expected Th2 deficient cellular response in IL-4Rα-/- and IL-33-/- mice compared to its genetic background BALB/c strain. Together, these data suggest that alterations in the Th1/Th2 balance or a complete ablation of Th1/Th2 responses can lead to major alterations in gut microbiota composition and function. Given the similarities between the human and mouse immune systems and gut microbiota, our finding that immune status is a strong driver of gut microbiota composition has important consequences for human immunodeficiency studies.

Activation of the Carboxypeptidase U (CPU, TAFIa, CPB2) System in Patients with SARS-CoV-2 Infection Could Contribute to COVID-19 Hypofibrinolytic State and Disease Severity Prognosis.

Coronavirus disease 2019 (COVID-19) is a viral lower respiratory tract infection caused by the highly transmissible and pathogenic SARS-CoV-2 (severe acute respiratory-syndrome coronavirus-2). Besides respiratory failure, systemic thromboembolic complications are frequent in COVID-19 patients and suggested to be the result of a dysregulation of the hemostatic balance. Although several markers of coagulation and fibrinolysis have been studied extensively, little is known about the effect of SARS-CoV-2 infection on the potent antifibrinolytic enzyme carboxypeptidase U (CPU). Blood was collected longitudinally from 56 hospitalized COVID-19 patients and 32 healthy controls. Procarboxypeptidase U (proCPU) levels and total active and inactivated CPU (CPU+CPUi) antigen levels were measured. At study inclusion (shortly after hospital admission), proCPU levels were significantly lower and CPU+CPUi antigen levels significantly higher in COVID-19 patients compared to controls. Both proCPU and CPU+CPUi antigen levels showed a subsequent progressive increase in these patients. Hereafter, proCPU levels decreased and patients were, at discharge, comparable to the controls. CPU+CPUi antigen levels at discharge were still higher compared to controls. Baseline CPU+CPUi antigen levels (shortly after hospital admission) correlated with disease severity and the duration of hospitalization. In conclusion, CPU generation with concomitant proCPU consumption during early SARS-CoV-2 infection will (at least partly) contribute to the hypofibrinolytic state observed in COVID-19 patients, thus enlarging their risk for thrombosis. Moreover, given the association between CPU+CPUi antigen levels and both disease severity and duration of hospitalization, this parameter may be a potential biomarker with prognostic value in SARS-CoV-2 infection.

Proline-specific peptidase activities (DPP4, PRCP, FAP and PREP) in plasma of hospitalized COVID-19 patients.

BACKGROUND: COVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. METHODS: Fifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. RESULTS: Compared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. CONCLUSION: The changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.

Predictive model for BNT162b2 vaccine response in cancer patients based on blood cytokines and growth factors.

Background: Patients with cancer, especially hematological cancer, are at increased risk for breakthrough COVID-19 infection. So far, a predictive biomarker that can assess compromised vaccine-induced anti-SARS-CoV-2 immunity in cancer patients has not been proposed. Methods: We employed machine learning approaches to identify a biomarker signature based on blood cytokines, chemokines, and immune- and non-immune-related growth factors linked to vaccine immunogenicity in 199 cancer patients receiving the BNT162b2 vaccine. Results: C-reactive protein (general marker of inflammation), interleukin (IL)-15 (a pro-inflammatory cytokine), IL-18 (interferon-gamma inducing factor), and placental growth factor (an angiogenic cytokine) correctly classified patients with a diminished vaccine response assessed at day 49 with >80% accuracy. Amongst these, CRP showed the highest predictive value for poor response to vaccine administration. Importantly, this unique signature of vaccine response was present at different studied timepoints both before and after vaccination and was not majorly affected by different anti-cancer treatments. Conclusion: We propose a blood-based signature of cytokines and growth factors that can be employed in identifying cancer patients at persistent high risk of COVID-19 despite vaccination with BNT162b2. Our data also suggest that such a signature may reflect the inherent immunological constitution of some cancer patients who are refractive to immunotherapy.

The Secretome of Filarial Nematodes and Its Role in Host-Parasite Interactions and Pathogenicity in Onchocerciasis-Associated Epilepsy.

Filarial nematodes secrete bioactive molecules which are of interest as potential mediators for manipulating host biology, as they are readily available at the host-parasite interface. The adult parasites can survive for years in the mammalian host, due to their successful modulation of the host immune system and most of these immunomodulatory strategies are based on soluble mediators excreted by the parasite. The secretome of filarial nematodes is a key player in both infection and pathology, making them an interesting target for further investigation. This review summarises the current knowledge regarding the components of the excretory-secretory products (ESPs) of filarial parasites and their bioactive functions in the human host. In addition, the pathogenic potential of the identified components, which are mostly proteins, in the pathophysiology of onchocerciasis-associated epilepsy is discussed.

Neuropathological Changes in Nakalanga Syndrome-A Case Report.

Nakalanga syndrome is a clinical manifestation of onchocerciasis-associated epilepsy characterized by stunting, delayed or absent secondary sexual development and skeletal deformities, and is often accompanied by epileptic seizures. The pathophysiology of Nakalanga syndrome is unknown. Here, we describe the post-mortem findings of a 17-year-old female who died with Nakalanga syndrome in northern Uganda. Macroscopic and histopathological examination of all major organs (liver, lungs, kidney and heart), including the brain and the pituitary gland, was performed. The suspected cause of death was malaria, and all major organs and pituitary gland appeared normal, except the lungs, which were edematous consistent with the malaria. Neuropathological changes include signs of neuro-inflammation (gliosis and activated microglia), which co-localized with tau-reactive neurofibrillary tangles and threads. The pathology was most abundant in the frontal cortex, thalamic and hypothalamic regions, and mesencephalon. The choroid plexus showed psammoma bodies. These findings indicate accelerated aging, probably due to repeated seizures. The neuropathological findings were similar to other persons who died with onchocerciasis-associated epilepsy. Examination of the pituitary gland did not reveal new information concerning the underlying pathophysiological mechanism of Nakalanga syndrome. Therefore, more post-mortem studies should be performed.

Potential Parasitic Causes of Epilepsy in an Onchocerciasis Endemic Area in the Ituri Province, Democratic Republic of Congo.

A high burden of epilepsy is observed in Africa where parasitological infections are endemic. In 2016, in an Onchocerciasis endemic area in the Logo health zone, in Ituri province in the Democratic Republic of Congo, a door-to-door study showed an epilepsy prevalence of 4.6%, and 50.6% of persons with epilepsy were infected with Onchocerca volvulus. In the current study, the serum of 195 people infected with O. volvulus persons with epilepsy were tested to determine the proportion of co-infections with Taenia solium, Toxocara canis and Strongyloides. These proportions were, respectively, 8.2, 18.5 and 12.8%. Persons with a T. solium co-infection were older than those without co-infection (p = 0.021). In six (37.5%) of the T. solium co-infected persons, the first seizures appeared after the age of 30 years compared to three (2.1%) persons without a co-infection (p < 0.0001). Our study suggests that an O. volvulus infection is the main parasitic cause of epilepsy in the Ituri province, but in some persons, mainly in those with late onset epilepsy and with focal seizures, the epilepsy may be caused by neurocysticercosis. As the population in the area rears pigs, activities to limit T. solium transmission should be implemented.

Cytokines and Onchocerciasis-Associated Epilepsy, a Pilot Study and Review of the Literature.

Neuro-inflammation may be associated with onchocerciasis-associated epilepsy (OAE) but thus far very few immunological studies have been performed in children with this form of epilepsy. In a pilot study we measured the cytokine levels in cerebrospinal fluid (CSF) of persons with OAE from Maridi, South Sudan, and from Mosango, Democratic Republic of the Congo (DRC) and compared these results with cytokine levels in CSF of Africans with non-OAE neurological disorders, and Europeans with epilepsy or other neurological conditions. The following cytokines were studied: IL-6, TNF-α, IL1-ß, IL-5, IL-4, IL-13, CCL3 (Mip-1α), VEGF-C, VCAM-1. No cytokine was significantly associated with OAE, although a lower IL-13 level was observed in CSF of persons with OAE compared to African controls. Observed cytokine profiles and neuro-inflammation may be the consequence of long-standing epilepsy, concomitant infections and malnutrition. Ideally cytokine levels should be determined in a prospective study in serum and CSF collected at the time of onset of the first seizures.

Onchocerca volvulus and epilepsy: A comprehensive review using the Bradford Hill criteria for causation.

BACKGROUND: The possibility that onchocerciasis may cause epilepsy has been suggested for a long time, but thus far, an etiological link has not been universally accepted. The objective of this review is to critically appraise the relationship between Onchocerca volvulus and epilepsy and subsequently apply the Bradford Hill criteria to further evaluate the likelihood of a causal association. METHODS: PubMed and gray literature published until September 15, 2020, were searched and findings from original research were synthesized. Adherence to the 9 Bradford Hill criteria in the context of onchocerciasis and epilepsy was determined to assess whether the criteria are met to strengthen the evidence base for a causal link between infection with O. volvulus and epilepsy, including the nodding syndrome. RESULTS: Onchocerciasis as a risk factor for epilepsy meets the following Bradford Hill criteria for causality: strength of the association, consistency, temporality, and biological gradient. There is weaker evidence supporting causality based on the specificity, plausibility, coherence, and analogy criteria. There is little experimental evidence. Considering the Bradford Hill criteria, available data suggest that under certain conditions (high microfilarial load, timing of infection, and perhaps genetic predisposition), onchocerciasis is likely to cause epilepsy including nodding and Nakalanga syndromes. CONCLUSION: Applying the Bradford Hill criteria suggests consistent epidemiological evidence that O. volvulus infection is a trigger of epilepsy. However, the pathophysiological mechanisms responsible for seizure induction still need to be elucidated.

Onchocerciasis Prevalence among Persons with Epilepsy in an Onchocerciasis Hypo-Endemic Area in the Democratic Republic of Congo: A Cross-Sectional Study.

A high epilepsy prevalence has been reported in onchocerciasis meso- and hyper-endemic regions in sub-Saharan Africa, including in the Democratic Republic of Congo (DRC). We investigated whether onchocerciasis-associated epilepsy can also be suspected in onchocerciasis hypo-endemic regions. Stored serum samples from 342 patients admitted with recent onset neurological symptoms admitted to Mosango general hospital, in the Kwilu province, DRC, between 2012 and 2015 were screened for onchocerciasis (OV16) antibodies by ELISA and Taenia solium antigen (using an in-house B158/B60 antigen test). Eighty-one (23.7%; 95% CI 19.5-28.5%) of these samples were positive for OV16 antibodies and 43/340 (12.6%; 95% CI 9.5-16.6%) were positive for T. solium antigen. Of the 58 persons clinically diagnosed with late onset epilepsy of unknown etiology, 19 (32.8%) were OV16 positive and nine (16%) T. solium antigen positive. In total, 16 persons with epilepsy were OV16 positive and T. solium negative, of whom 12 (75%) were between the ages seven to 31 years old, an age rage in which onchocerciasis-associated epilepsy is observed. Our study suggests that in onchocerciasis hypo-endemic areas, in T. solium antigen negative persons with epilepsy, onchocerciasis should be considered as a potential trigger of epilepsy.