Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

Full-length MSP1 is a major target of protective immunity after controlled human malaria infection.

The merozoite surface protein 1 (MSP1) is the most abundant protein on the surface of the invasive merozoite stages of Plasmodium falciparum and has long been considered a key target of protective immunity. We used samples from a single controlled human malaria challenge study to test whether the full-length version of MSP1 (MSP1FL) induced antibodies that mediated Fc-IgG functional activity in five independent assays. We found that anti-MSP1FL antibodies induced complement fixation via C1q, monocyte-mediated phagocytosis, neutrophil respiratory burst, and natural killer cell degranulation as well as IFNγ production. Activity in each of these assays was strongly associated with protection. The breadth of MSP1-specific Fc-mediated effector functions was more strongly associated with protection than the individual measures and closely mirrored what we have previously reported using the same assays against merozoites. Our findings suggest that MSP1FL is an important target of functional antibodies that contribute to a protective immune response against malaria.

Doxycycline for the treatment of nodding syndrome: a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Nodding syndrome is a poorly understood neurological disorder that predominantly occurs in Africa. We hypothesised that nodding syndrome is a neuroinflammatory disorder, induced by antibodies to Onchocerca volvulus or its Wolbachia symbiont, cross-reacting with host neuronal proteins (HNPs), and that doxycycline can be used as treatment. METHODS: In this randomised, double-blind, placebo-controlled, phase 2 trial, we recruited participants from districts affected by nodding syndrome in northern Uganda. We included children and adolescents aged 8-18 years with nodding syndrome, as defined by WHO consensus criteria. Participants were randomly assigned (1:1) to receive either 100 mg doxycycline daily or placebo for 6 weeks via a computer-generated schedule stratified by skin microscopy results, and all parties were masked to group assignment. Diagnoses of O volvulus and antibodies to HNPs were made using luciferase immunoprecipitation system assays and immunohistochemistry. The primary outcome was change in the proportion with antibodies to HNPs, assessed at 24 months. All participants were included in safety analyses, and surviving participants (those with samples at 24 months) were included in primary analyses. Secondary outcomes were: change in concentrations of antibodies to HNPs at 24 months compared with baseline; proportion of participants testing positive for antibodies to O volvulus-specific proteins and concentrations of Ov16 or OVOC3261 antibodies at 24 months compared with baseline; change in seizure burden, proportion achieving seizure freedom, and the proportions with interictal epileptiform discharges on the diagnostic EEG; overall quality of life; disease severity at 24 months; and incidence of all-cause adverse events, serious adverse events, and seizure-related mortality by 24 months. This trial is registered with ClinicalTrials.gov, NCT02850913. FINDINGS: Between Sept 1, 2016, and Aug 31, 2018, 329 children and adolescents were screened, of whom 240 were included in the study. 140 (58%) participants were boys and 100 (42%) were girls. 120 (50%) participants were allocated to receive doxycycline and 120 (50%) to receive placebo. At recruitment, the median duration of symptoms was 9 years (IQR 6-10); 232 (97%) participants had O volvulus-specific antibodies and 157 (65%) had autoantibodies to HNPs. The most common plasma autoantibodies were to human protein deglycase DJ-1 (85 [35%] participants) and leiomodin-1 (77 [32%] participants) and, in cerebrospinal fluid (CSF), to human DJ-1 (27 [11%] participants) and leiomodin-1 (14 [6%] participants). On immunohistochemistry, 46 (19%) participants had CSF autoantibodies to HNPs, including leiomodin-1 (26 [11%]), γ-aminobutyric acid B receptors (two [<1%]), CASPR2 (one [<1%]), or unknown targets (28 [12%]). At 24 months, 161 (72%) of 225 participants had antibodies to HNPs compared with 157 (65%) of 240 at baseline. 6 weeks of doxycycline did not affect the concentration of autoantibodies to HNPs, seizure control, disease severity, or quality of life at the 24-month follow-up but substantially decreased Ov16 antibody concentrations; the median plasma signal-to-noise Ov16 ratio was 16·4 (95% CI 6·4-38·4), compared with 27·9 (8·2-65·8; p=0·033) for placebo. 14 (6%) participants died and, other than one traffic death, all deaths were seizure-related. Acute seizure-related hospitalisations (rate ratio [RR] 0·43 [95% CI 0·20-0·94], p=0·028) and deaths (RR 0·46 [0·24-0·89], p=0·028) were significantly lower in the doxycycline group. At 24 months, 96 (84%) of 114 participants who received doxycycline tested positive for antibodies to Ov16, compared with 97 (87%) of 111 on placebo (p=0·50), and 74 (65%) participants on doxycycline tested positive for antibodies to OVOC3261, compared with 57 (51%) on placebo (p=0·039). Doxycycline was safe; there was no difference in the incidence of grade 3-5 adverse events across the two groups. INTERPRETATION: Nodding syndrome is strongly associated with O volvulus and the pathogenesis is probably mediated through an O volvulus induced autoantibody response to multiple proteins. Although it did not reverse disease symptoms, doxycycline or another prophylactic antibiotic could be considered as adjunct therapy to antiseizure medication, as it might reduce fatal complications from acute seizures and status epilepticus induced by febrile infections. FUNDING: Medical Research Council (UK). TRANSLATION: For the Luo translation of the abstract see Supplementary Materials section.

Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

Antibody-Dependent Respiratory Burst against Plasmodium falciparum Merozoites in Individuals Living in an Area with Declining Malaria Transmission.

Malaria transmission intensity affects the development of naturally acquired immunity to malaria. An absolute correlate measure of protection against malaria is lacking. However, antibody-mediated functions against Plasmodium falciparum correlate with protection against malaria. In children, antibody-mediated functions against P. falciparum decline with reduced exposure. It is unclear whether adults maintain antibody-mediated functions as malaria transmission declines. This study assessed antibody-dependent respiratory burst (ADRB) in individuals from an area with declining malaria transmission. In an age-matched analysis, we compare ADRB activity during high versus low malaria transmission periods. Age significantly predicted higher ADRB activity in the high (p < 0.001) and low (p < 0.001) malaria transmission periods. ADRB activity was higher during the high compared to the low malaria transmission period in older children and adults. Only older adults during the high malaria transmission period had their median ADRB activity above the ADRB cut-off. Ongoing P. falciparum infection influenced ADRB activity during the low (p = 0.01) but not the high (p = 0.29) malaria transmission period. These findings propose that naturally acquired immunity to P. falciparum is affected in children and adults as malaria transmission declines, implying that vaccines will be necessary to induce and maintain protection against malaria.

Comparison of Structural Changes in Nodding Syndrome and Other Epilepsies Associated With Onchocerca volvulus.

BACKGROUND AND OBJECTIVE: Nodding syndrome (NS) is a unique childhood-onset epileptic disorder that occurs predominantly in several regions of sub-Saharan Africa. The disease has been associated with Onchocerca volvulus (Ov)-induced immune responses and possible cross-reactivity with host proteins. The aim of this study was to compare structural changes in the brain on MRI between NS and other forms of onchocerciasis-associated epilepsies (OAEs) and to relate structural changes to the Ov-induced immune responses and level of disability. METHODS: Thirty-nine children with NS and 14 age-matched participants with other forms of OAE from an endemic region in Uganda underwent detailed clinical examination, serologic evaluation (including Ov-associated antibodies to Ov-16 and Hu-leiomodin-1) and quantitative volumetric analysis of brain MRIs (1.5 T scanner) using Neuroreader, a cloud-based software. RESULTS: Cerebral and cerebellar atrophy were the predominant features in both NS and OAE. On quantitative volumetric analysis, participants with NS had larger ventricular volumes compared with participants with OAE, indicative of increased global cortical atrophy (pcorr = 0.036). Among children with NS, severe disability correlated with higher degree of atrophy in the gray matter volume (pcorr = 0.009) and cerebellar volume (pcorr = 0.009). NS cases had lower anti-Ov-16 IgG signal-to-noise ratios than the OAE cases (p < 0.01), but no difference in the levels of the Hu-leiomodin-1 antibodies (p = 0.64). The levels of Ov-associated antibodies did not relate to the degree of cerebral or cerebellar atrophy in either NS or OAE cases. DISCUSSION: This is the first study to show that cerebral and cerebellar atrophy correlated with the severity of NS disability, providing an imaging marker for these endemic epileptic disorders that until now have remained poorly characterized. Both NS and OAE have cerebral and cerebellar atrophy, and the levels of Ov-associated antibodies do not seem to be related to the structural changes on MRI.

Asplenia and spleen hypofunction.

Asplenia (the congenital or acquired absence of the spleen) and hyposplenism (defective spleen function) are common causes of morbidity and mortality. The spleen is a secondary lymphoid organ that is responsible for the regulation of immune responses and blood filtration. Hence, asplenia or hyposplenism increases susceptibility to severe and invasive infections, especially those sustained by encapsulated bacteria (namely, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b). Asplenia is predominantly due to splenectomy for either traumatic events or oncohaematological conditions. Hyposplenism can be caused by several conditions, including haematological, infectious, autoimmune and gastrointestinal disorders. Anatomical disruption of the spleen and depletion of immune cells, especially IgM memory B cells, seem to be predominantly responsible for the clinical manifestations. Early recognition of hyposplenism and proper management of asplenia are warranted to prevent overwhelming post-splenectomy infections through vaccination and antibiotic prophylaxis. Although recommendations are available, the implementation of vaccination strategies, including more effective and immunogenic vaccines, is needed. Additionally, screening programmes for early detection of hyposplenism in high-risk patients and improvement of patient education are warranted.

Neutrophil gelatinase-associated lipocalin is elevated in children with acute kidney injury and sickle cell anemia, and predicts mortality.

Urine neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker of acute kidney injury that has been adapted to a urine dipstick test. However, there is limited data on its use in low-and-middle-income countries where diagnosis of acute kidney injury remains a challenge. To study this, we prospectively enrolled 250 children with sickle cell anemia aged two to 18 years encompassing 185 children hospitalized with a vaso-occlusive pain crisis and a reference group of 65 children attending the sickle cell clinic for routine care follow up. Kidney injury was defined using serial creatinine measures and a modified-Kidney Disease Improving Global Outcome definition for sickle cell anemia. Urine NGAL was measured using the NGAL dipstick and a laboratory reference. The mean age of children enrolled was 8.9 years and 42.8% were female. Among hospitalized children, 36.2% had kidney injury and 3.2% died. Measured urine NGAL levels by the dipstick were strongly correlated with the standard enzyme-linked immunosorbent assay for urine NGAL (hospitalized children, 0.71; routine care reference, 0.88). NGAL levels were elevated in kidney injury and significantly increased across injury stages. Hospitalized children with a high-risk dipstick test (300ng/mL and more) had a 2.47-fold relative risk of kidney injury (95% confidence interval 1.68 to 3.61) and 7.28 increased risk of death (95% confidence interval 1.10 to 26.81) adjusting for age and sex. Thus, urine NGAL levels were found to be significantly elevated in children with sickle cell anemia and acute kidney injury and may predict mortality.

Epstein-Barr virus and malaria upregulate AID and APOBEC3 enzymes, but only AID seems to play a major mutagenic role in Burkitt lymphoma.

Endemic Burkitt lymphoma (eBL) is characterized by an oncogenic IGH/c-MYC translocation and Epstein-Barr virus (EBV) positivity, and is epidemiologically linked to Plasmodium falciparum malaria. Both EBV and malaria are thought to contribute to eBL by inducing the expression of activation-induced cytidine deaminase (AID), an enzyme involved in the IGH/c-MYC translocation. AID/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) family enzymes have recently emerged as potent mutagenic sources in a variety of cancers, but apart from AID, their involvement in eBL and their regulation by EBV and P. falciparum is unknown. Here, we show that upon inoculation with EBV, human B cells strongly upregulate the expression of enzymatically active APOBEC3B and APOBEC3G. In addition, we found significantly increased levels of APOBEC3A in B cells of malaria patients, which correlated with parasite load. Interestingly, despite the fact that APOBEC3A, APOBEC3B, and APOBEC3G caused c-MYC mutations when overexpressed in HEK293T cells, a mutational enrichment in eBL tumors was only detected in AID motifs. This suggests that even though the EBV- and P. falciparum-directed immune response triggers the expression and activity of several AID/APOBEC members, only the upregulation of AID has oncogenic consequences, while the induction of the APOBEC3 subfamily may primarily have immunoprotective functions.

Neuroimmunology of Common Parasitic Infections in Africa.

Parasitic infections of the central nervous system are an important cause of morbidity and mortality in Africa. The neurological, cognitive, and psychiatric sequelae of these infections result from a complex interplay between the parasites and the host inflammatory response. Here we review some of the diseases caused by selected parasitic organisms known to infect the nervous system including Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei spp., and Taenia solium species. For each parasite, we describe the geographical distribution, prevalence, life cycle, and typical clinical symptoms of infection and pathogenesis. We pay particular attention to how the parasites infect the brain and the interaction between each organism and the host immune system. We describe how an understanding of these processes may guide optimal diagnostic and therapeutic strategies to treat these disorders. Finally, we highlight current gaps in our understanding of disease pathophysiology and call for increased interrogation of these often-neglected disorders of the nervous system.

Characterization of Anopheles gambiae D7 salivary proteins as markers of human-mosquito bite contact.

BACKGROUND: Malaria is transmitted when infected Anopheles mosquitoes take a blood meal. During this process, the mosquitoes inject a cocktail of bioactive proteins that elicit antibody responses in humans and could be used as biomarkers of exposure to mosquito bites. This study evaluated the utility of IgG responses to members of the Anopheles gambiae D7 protein family as serological markers of human-vector contact. METHODS: The D7L2, D7r1, D7r2, D7r3, D7r4 and SG6 salivary proteins from An. gambiae were expressed as recombinant antigens in Escherichia coli. Antibody responses to the salivary proteins were compared in Europeans with no prior exposure to malaria and lifelong residents of Junju in Kenya and Kitgum in Uganda where the intensity of malaria transmission is moderate and high, respectively. In addition, to evaluate the feasibility of using anti-D7 IgG responses as a tool to evaluate the impact of vector control interventions, we compared responses between individuals using insecticide-treated bednets to those who did not in Junju, Kenya where bednet data were available. RESULTS: We show that both the long and short forms of the D7 salivary gland antigens elicit a strong antibody response in humans. IgG responses against the D7 antigens reflected the transmission intensities of the three study areas, with the highest to lowest responses observed in Kitgum (northern Uganda), Junju (Kenya) and malaria-naïve Europeans, respectively. Specifically, the long form D7L2 induced an IgG antibody response that increased with age and that was lower in individuals who slept under a bednet, indicating its potential as a serological tool for estimating human-vector contact and monitoring the effectiveness of vector control interventions. CONCLUSIONS: This study reveals that D7L2 salivary antigen has great potential as a biomarker of exposure to mosquito bites and as a tool for assessing the efficacy of vector control strategies such as bednet use.

Risk Factors for Nodding Syndrome and Other Forms of Epilepsy in Northern Uganda: A Case-Control Study.

Epidemiological studies suggest a link between onchocerciasis and various forms of epilepsy, including nodding syndrome (NS). The aetiopathology of onchocerciasis associated epilepsy remains unknown. This case-control study investigated potential risk factors that may lead to NS and other forms of non-nodding epilepsy (OFE) in northern Uganda. We consecutively recruited 154 persons with NS (aged between 8 and 20 years), and age-frequency matched them with 154 with OFE and 154 healthy community controls. Participants' socio-demography, medical, family, and migration histories were recorded. We tested participants for O. volvulus serum antibodies. The 154 controls were used for both OFE and NS separately to determine associations. We recruited 462 people with a median age of 15 years (IQR 14, 17); 260 (56.4%) were males. Independent risk factors associated with the development of NS were the presence of O. volvulus antibodies [aOR 8.79, 95% CI (4.15-18.65), p-value < 0.001] and preterm birth [aOR 2.54, 95% CI (1.02-6.33), p-value = 0.046]. Risk factors for developing OFE were the presence of O. volvulus antibodies [aOR 8.83, 95% CI (4.48-17.86), p-value < 0.001] and being born in the period before migration to IDP camps [aOR 4.28, 95% CI (1.20-15.15), p-value = 0.024]. In conclusion, O. volvulus seropositivity was a risk factor to develop NS and OFE; premature birth was a potential co-factor. Living in IDP camps was not a risk factor for developing NS or OFE.

Epilepsy in Onchocerca volvulus Sero-Positive Patients From Northern Uganda-Clinical, EEG and Brain Imaging Features.

Globally, epilepsy is the most common chronic neurological disorder. The incidence in sub-Saharan Africa is 2-3 times higher than that in high income countries. Infection by Onchocerca volvulus may be an underlying risk factor for the high burden and based upon epidemiological associations, has been proposed to cause a group of disorders-Onchocerca associated epilepsies (OAE) like nodding syndrome (NS). To improve our understanding of the disease spectrum, we described the clinical, electroencephalographic (EEG) and magnetic resonance imaging (MRI) features of children with epilepsy and sero-positive for Onchocerca volvulus (possible OAEs other than nodding syndrome). Twenty-nine children and adolescents with non-nodding syndrome OAE in northern Uganda were enrolled. A diagnosis of OAE was made in patients with epilepsy and seizure onset after age 3 years, no reported exposure to perinatal severe febrile illness or traumatic brain injury, no syndromic epilepsy diagnosis and a positive Ov-16 ELISA test. Detailed clinical evaluation including psychiatric, diagnostic EEG, a diagnostic brain MRI (in 10 patients) and laboratory testing were performed. Twenty participants (69%) were male. The mean age was 15.9 (standard deviation [SD] 1.9) years while the mean age at seizure onset was 9.8 (SD 2.9) years. All reported normal early childhood development. The most common clinical presentation was a tonic-clonic seizure. The median number of seizures was 2 (IQR 1-4) in the previous month. No specific musculoskeletal changes, or cranial nerve palsies were reported, neither were any vision, hearing and speech difficulties observed. The interictal EEG was abnormal in the majority with slow wave background activity in 52% (15/29) while 41% (12/29) had focal epileptiform activity. The brain MRI showed mild to moderate cerebellar atrophy and varying degrees of atrophy of the frontal, parietal and occipital lobes. The clinical spectrum of epilepsies associated with Onchocerca may be broader than previously described. In addition, focal onset tonic-clonic seizures, cortical and cerebellar atrophy may be important brain imaging and clinical features.

Systemic and cerebrospinal fluid immune and complement activation in Ugandan children and adolescents with long-standing nodding syndrome: A case-control study.

OBJECTIVE: Nodding syndrome is a poorly understood epileptic encephalopathy characterized by a unique seizure type-head nodding-and associated with Onchocerca volvulus infection. We hypothesized that altered immune activation in the cerebrospinal fluid (CSF) and plasma of children with nodding syndrome may yield insights into the pathophysiology and progression of this seizure disorder. METHOD: We conducted a case-control study of 154 children (8 years or older) with long-standing nodding syndrome and 154 healthy age-matched community controls in 3 districts of northern Uganda affected by nodding syndrome. Control CSF samples were obtained from Ugandan children in remission from hematological malignancy during routine follow-up. Markers of immune activation and inflammation (cytokines and chemokines) and complement activation (C5a) were measured in plasma and CSF using ELISA or Multiplex Luminex assays. O volvulus infection was assessed by serology for anti-OV-16 IgG levels. RESULTS: The mean (SD) age of the population was 15.1 (SD: 1.9) years, and the mean duration of nodding syndrome from diagnosis to enrollment was 8.3 (SD: 2.7) years. The majority with nodding syndrome had been exposed to O volvulus (147/154 (95.4%)) compared with community children (86/154 (55.8%)), with an OR of 17.04 (95% CI: 7.33, 45.58), P < .001. C5a was elevated in CSF of children with nodding syndrome compared to controls (P < .0001). The levels of other CSF markers tested were comparable between cases and controls after adjusting for multiple comparisons. Children with nodding syndrome had lower plasma levels of IL-10, APRIL, CCL5 (RANTES), CCL2, CXCL13, and MMP-9 compared with community controls (P < .05 for all; multiple comparisons). Plasma CRP was elevated in children with nodding syndrome compared to community children and correlated with disease severity. SIGNIFICANCE: Nodding syndrome is associated with exposure to O. volvulus. Compared to controls, children with long-standing symptoms of nodding syndrome show evidence of complement activation in CSF and altered immune activation in plasma.

Household poverty, schooling, stigma and quality of life in adolescents with epilepsy in rural Uganda.

BACKGROUND: Epilepsy remains a leading chronic neurological disorder in Low- and Middle-Income Countries. In Uganda, the highest burden is among young rural people. We aimed to; (i) describe socio-economic status (including schooling), and household poverty in adolescents living with epilepsy (ALE) compared to unaffected counterparts in the same communities and (ii) determine the factors associated with the overall quality of life (QoL). METHODS: This was a cross-sectional survey nested within a larger study of ALE compared to age-matched healthy community children in Uganda. Between Sept 2016 to Sept 2017, 154 ALE and 154 healthy community controls were consecutively recruited. Adolescents recruited were frequency and age-matched based on age categories 10-14 and 15-19 years. Clinical history and standardized assessments were conducted. One control participant had incomplete assessment and was excluded. The primary outcome was overall QoL and key variables assessed were schooling status and household poverty. Descriptive and multivariable linear regression analysis were conducted for independent associations with overall QoL. RESULTS: Mean (SD) age at seizure onset was 8.8 (3.9) years and median (IQR) monthly seizure burden was 2 (1-4). Epilepsy was associated with living in homes with high household poverty; 95/154 (61.7%) ALE lived in the poorest homes compared to 68/153 (44.5%) of the healthy adolescents, p = 0.001. Nearly two-thirds of ALE had dropped out of school and only 48/154 (31.2%) were currently attending school compared to 136/153 (88.9%) of healthy controls, p < 0.001. QoL was lowest among ALE who never attended school (p < 0.001), with primary education (p = 0.006) compared to those with at least secondary education. Stigma scores [mean(SD)] were highest among ALE in the poorest [69.1(34.6)], and wealthy [70.2(32.2)] quintiles compared to their counterparts in poorer [61.8(31.7)], medium [68.0(32.7)] and wealthiest [61.5(33.3)] quintiles, though not statistically significant (p = 0.75). After adjusting for covariates, ALE currently attending school had higher overall QoL compared to their counterparts who never attended school (ß = 4.20, 95%CI: 0.90,7.49, p = 0.013). QoL scores were higher among ALE with ≥secondary education than those with no or primary education (ß = 10.69, 95%CI: 1.65, 19.72). CONCLUSIONS: ALE in this rural area are from the poorest households, are more likely to drop out of school and have the lowest QoL. Those with poorer seizure control are most affected. ALE should be included among vulnerable population groups and in addition to schooling, strategies for seizure control and addressing the epilepsy treatment gap in affected homes should be specifically targeted in state poverty eradication programs.

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.

Doxycycline for the treatment of nodding syndrome (DONS); the study protocol of a phase II randomised controlled trial.

BACKGROUND: Nodding syndrome is a poorly understood neurological disorder of unknown aetiology, affecting several thousand children in Africa. There has been a consistent epidemiological association with infection by the filarial parasite, Onchocerca volvulus and antibodies to leiomodin and DJ-1, cross-reacting with O.volvulus proteins, have been reported. We hypothesized that nodding syndrome is a neuro-inflammatory disorder, induced by antibodies to O.volvulus or its symbiont, Wolbachia, cross-reacting with human neuron proteins and that doxycycline, which kills Onchocerca through effects on Wolbachia, may be used as treatment. METHODS: This will be a two-arm, double-blind, placebo-controlled, randomised phase II trial of doxycycline 100 mg daily for six weeks in 230 participants. Participants will be patients' ages≥8 years with nodding syndrome. They will receive standard of care supportive treatment. All will be hospitalised for 1-2 weeks during which time baseline measurements including clinical assessments, EEG, cognitive and laboratory testing will be performed and antiepileptic drug doses rationalised. Participants will then be randomised to either oral doxycycline (Azudox®, Kampala Pharmaceutical Industries) 100 mg daily or placebo. Treatment will be initiated in hospital and continued at home. Participants will be visited at home at 2, 4 and 6 weeks for adherence monitoring. Study outcomes will be assessed at 6, 12, 18 and 24-month visits. Analysis will be by intention to treat. The primary efficacy outcome measure will be the proportion of patients testing positive and the levels or titires of antibodies to host neuron proteins (HNPs) and/or leiomodin at 24 months. Secondary outcome measures will include effect of the intervention on seizure control, inflammatory markers, cognitive function, disease severity and quality of life. DISCUSSION: This trial postulates that targeting O.volvulus through drugs which kill Wolbachia can modify the pathogenic processes in nodding syndrome and improve outcomes. Findings from this study are expected to substantially improve the understanding and treatment of nodding syndrome. TRIAL REGISTRATION: Registered with clinicaltrials.gov ID: NCT02850913 on 1st August, 2016.

The natural history of nodding syndrome.

Nodding syndrome is a poorly understood acquired disorder affecting children in sub-Saharan Africa. The aetiology and pathogenesis are unknown, and no specific treatment is available. Affected children have a distinctive feature (repeated clusters of head nodding) and progressively develop many other features. In an earlier pilot study, we proposed a five-level clinical staging system. The present study aimed to describe the early features and natural history of nodding syndrome and refine the proposed clinical stages. This was a retrospective study of the progressive development of symptoms and complications of nodding syndrome. Participants were a cohort of patients who had been identified by community health workers and were referred for treatment. A detailed history was obtained to document the chronological development of symptoms before and after onset of head nodding and a physical examination and disability assessment performed by a team of clinicians and therapists. A total of 210 children were recruited. The mean age at the onset of head nodding was 7.5 (SD: 3.0) years. Five overlapping clinical stages were recognised: prodromal, head nodding, convulsive seizures, multiple impairments, and severe disability stages. Clinical features before the onset of head nodding (prodromal features) included periods of staring blankly or being inattentive, complaints of dizziness, excessive sleepiness, lethargy, and general body weakness, all occurring two weeks to 24 months before nodding developed. After the onset of head nodding, patients progressively developed convulsive seizures, cognitive and psychiatric dysfunction, physical deformities, growth arrest, and eventually, in some patients, severe disability. The description of the natural history of nodding syndrome and especially the prodromal features has the potential of providing a means for the early identification of at-risk patients and the prompt initiation of interventions before extensive brain injury develops. The wide spectrum of symptoms and complications emphasises the need for multi-disciplinary investigations and care.

High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories.

BACKGROUND: Malaria is still a major health problem in sub-Saharan Africa and south-east Asia, but research on malaria in low-income countries can be a challenge due to the lack of laboratory equipment. In addition, severe malaria mainly affects very young children, which limits the amount of blood available for research purposes. Thus, there is a need for protocols that yield a maximum of information from a minimum amount of blood, which are operable in basically equipped laboratories. RESULTS: A protocol for tandem B and T helper (Th) cell isolation directly from whole blood, and a freezer-independent sample preservation method compatible with the warm and humid climate of malaria regions was established and validated. The protocol thereby circumvents the need of high-technology centrifuges and unimpeachable power supply for peripheral blood mononuclear cell isolation. Both purity and yield are excellent. Depending on the expression level of the genes of interest, between 2 and 5 ml of blood are adequate for reliable qRT-PCR results from both B and Th cells of healthy paediatric donors as well as paediatric malaria patients. CONCLUSION: This protocol for high purity high yield B cell and Th cell isolation and sample storage for subsequent qRT-PCR analysis from a minimal amount of blood is contrivable with basic equipment and independent of continuous power supply. Thus, it is likely to be of avail for many scientists performing malaria research in rural institutes or hospitals, and thus in countries where malaria is most prevalent.

Asymptomatic malaria parasitaemia and seizure control in children with nodding syndrome; a cross-sectional study.

OBJECTIVE: Plasmodium falciparum is epileptogenic and in malaria endemic areas, is a leading cause of acute seizures. In these areas, asymptomatic infections are common but considered benign and so, are not treated. The effects of such infections on seizures in patients with epilepsy is unknown. This study examined the relationship between P. falciparum infection and seizure control in children with a unique epilepsy type, the nodding syndrome. DESIGN: This cross-sectional study was nested in an ongoing trial 'Doxycycline for the treatment of nodding syndrome (NCT02850913)'. We hypothesised that, in patients with epilepsy, infection by P. falciparum, including asymptomatic infections, increases the risk of seizures and impairs seizure control. SETTING AND PARTICIPANTS: Participants were Ugandan children with nodding syndrome, age ≥8 years, receiving sodium valproate. All had standardised testing including documentation of the number of seizures in the past month, a rapid malaria test and if positive, the peripheral blood parasite density. OUTCOMES: The primary outcome was the number of seizures in the past month (30 days). RESULTS: A total of 164/240 (68%) had malaria. Asymptomatic infections (without fever) were seen in 160/240 (67%) and symptomatic infections in 4/240 (2.7%). In participants without malaria, the median (IQR) number of seizures in the past month was 2.0 (1.0-4.0) and it was 4.0 (2.0-7.5) in participants with malaria, p=0.017. The number of seizures in asymptomatic persons was 3.0 (IQR 2.0-7.3) and 6.0 (IQR 4.0-10.0) in symptomatic individuals, p=0.024. Additionally, in asymptomatic patients, a positive correlation was observed between the parasite density and number of seizures, r=0.33, p=0.002. CONCLUSION: In patients with nodding syndrome, both asymptomatic and symptomatic malaria are associated with an increased risk of seizures and poorer seizure control. Similar effects should be examined in other epilepsy disorders. Malaria prevention should be strengthened for these patients and chemotreatment and prevention studies considered to improve seizure control.