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High propensity for multidrug-resistant pneumococcal shedding among adults living with HIV on stable antiretroviral therapy in Malawi
Abstract Background People living with HIV (PLHIV) on antiretroviral therapy (ART) are still at risk of pneumococcal disease and have over two-fold higher pneumococcal carriage prevalence than HIV-uninfected adults (HIV- adults). Carriage is a risk factor for pneumococcal disease, antimicrobial resistance (AMR) emergence, and transmission. Therefore, we tested whether the high prevalence of pneumococcal carriage in PLHIV on ART is associated with increased bacterial density, shedding and AMR. Methods We recruited asymptomatic PLHIV on ART for more than one year (PLHIV-ART>1yr) and HIV- adults. Nasopharyngeal swabs were collected on days 3, 7, 14, 21, and 28, followed by monthly collections for 12 months, while shedding samples were collected on days 3, 21, and 28. Peripheral blood was collected on day 3 to measure CD4 count and HIV viral load. Pneumococcal carriage density and shedding were assessed using standard bacterial culture, and multiple carriage was detected through whole plate sweep sequencing. AMR profiling was conducted using disk diffusion and E-test. Findings PLHIV-ART>1yr had a higher propensity for high-density carriage (adjusted Odds Ratio 1.67, 95% CI 1.07-2.60, p=0.023). Moreover, PLHIV-ART>1yr are more likely to shed pneumococci than HIV- adults (aOR 2.52, 95% CI 1.06-6.00, p=0.037), with carriage density identified as an important risk factor for shedding (aOR 3.35, 95% CI 1.55-7.24, p=0.002). Aerosol shed isolates from PLHIV-ART>1yr were mostly multidrug-resistant (62% 18/29, 95% CI 48%-77%). Interpretation These findings indicate that PLHIV-ART>1yr remain at high risk of pneumococcal disease and could also be an important reservoir for shedding multidrug-resistant pneumococci.
Electroencephalogram Features Distinguish Cases of Cerebral Malaria Among Malawian Children With Fever and Coma.
BACKGROUND: In febrile comatose patients living in malaria-endemic areas, overlapping symptoms and limited laboratory capacity make it difficult to distinguish parasitic, bacterial, and viral central nervous system infections. We evaluated electroencephalography (EEG) as a biomarker to differentiate the microbiologic etiology of pediatric febrile coma at a major referral center in Malawi. METHODS: This was a retrospective case-control study comparing EEG recordings of Malawian children with cerebral malaria to those with febrile coma of nonmalarial cause (bacterial meningitis, viral encephalitis, or unknown cause). Participants were admitted to Queen Elizabeth Central Hospital (Blantyre, Malawi) between 2013 and 2021. Inclusion criteria were fever, coma (Blantyre Coma Score ≤2), and coma etiology (malarial or nonmalarial) defined by laboratory testing. Four supervised machine learning algorithms were used to train a balanced ensemble classifier, SuperLearner, generating test characteristics of the diagnostic ability of EEG features. RESULTS: Two hundred three children with cerebral malaria and 87 children with nonmalarial coma were included. Univariate analysis of qualitative (visual) EEG interpretations revealed higher voltage, slower background frequency, more sleep elements, less variability, more abnormal organization, and less continuity in cerebral malaria. Quantitative waveform analysis showed greater power in cerebral malaria. Both quantitative and qualitative EEG interpretation distinguished coma etiology (area under the receiver operating characteristic curve [AUROC] = 0.85 and 0.86, respectively). Combining qualitative and quantitative interpretation methods, the test characteristic improved (AUROC = 0.90). CONCLUSIONS: EEG features distinguish malarial from nonmalarial coma in febrile Malawian children. This technology may aid in distinguishing the microbiologic etiology of febrile coma in malaria-endemic areas.
Using Electroencephalography to Assess Coma Etiology in Children with Retinopathy-Negative Cerebral Malaria.
Autopsy studies of children dying of cerebral malaria (CM) have revealed that those with malarial retinopathy exhibited high levels of sequestration in the cerebral vasculature, whereas children with retinopathy-negative CM exhibited lower sequestration levels and possible nonmalarial causes of death. This suggests that children dying of retinopathy-negative CM have nonmalarial coma etiologies with concomitant incidental parasitemia, which is common in high malaria transmission areas. Subsequent studies have challenged this assertion, positing that retinopathy-negative CM and retinopathy-positive CM are variants of the same disease pathophysiology or host biology, both caused by acute malaria infection. We recently determined that electroencephalography (EEG) can be used to discriminate between a malarial coma (CM) and a nonmalarial coma. To better understand the contribution of acute malaria infection in the pathophysiology of retinopathy-negative CM, we compared qualitative and quantitative EEG findings from 30-minute EEG recordings of Malawian children aged 3 months to 14 years hospitalized at Queen Elizabeth Central Hospital with retinopathy-negative CM, retinopathy-positive CM, and nonmalarial coma. Neither qualitative nor quantitative EEG interpretation methods allow for the discrimination between children with retinopathy-positive CM and those with retinopathy-negative CM. Conversely, quantitative EEG readily differentiated children with retinopathy-negative CM from those with nonmalarial coma (area under the receiving operating characteristic [AUROC] curve of 0.83). When combining qualitative and quantitative EEG interpretation methods, the ability of EEG to distinguish retinopathy-negative CM from nonmalarial EEG increases (AUROC of 0.87). The EEGs of children with retinopathy-negative CM are similar to those of children with retinopathy-positive CM and significantly different from those of children with nonmalarial coma, supporting the hypothesis that acute malarial infection is pathophysiologically important in retinopathy-negative CM.
Early Cardiac Dysfunction in Adolescents With Low Vigorous Physical Activity.
BACKGROUND: Adverse left ventricular diastolic function (LVDF) is an early marker of cardiac dysfunction that worsens with age and can lead to heart failure. It is unclear when this deterioration begins and whether physical activity (PA) influences it. We assessed the independent relationships of adverse LVDF in adolescents with different PA intensities, compared to the association with adiposity. The impact of adverse LVDF from low PA on cardiorespiratory fitness was examined. METHODS: In 127 adolescents (aged 11-18 years), we assessed LVDF by echocardiography, 7-day PA by wrist-worn accelerometry, adiposity Z scores by bioimpedance, and peak oxygen consumption by cardiopulmonary exercise testing (OxSOCRATES [Oxfordshire Sedentariness, Obesity, and Cardiometabolic Risk in Adolescents: A Trial of Exercise in Schools] study; NCT04118543). Adverse LVDF was defined using a body surface area-adjusted septal early diastolic tissue peak velocity Z score (
The molecular reach of antibodies crucially underpins their viral neutralisation capacity.
Key functions of antibodies, such as viral neutralisation, depend on high-affinity binding. However, viral neutralisation poorly correlates with antigen affinity for reasons that have been unclear. Here, we use a new mechanistic model of bivalent binding to study >45 patient-isolated IgG1 antibodies interacting with SARS-CoV-2 RBD surfaces. The model provides the standard monovalent affinity/kinetics and new bivalent parameters, including the molecular reach: the maximum antigen separation enabling bivalent binding. We find large variations in these parameters across antibodies, including reach variations (22-46 nm) that exceed the physical antibody size (~15 nm). By using antigens of different physical sizes, we show that these large molecular reaches are the result of both the antibody and antigen sizes. Although viral neutralisation correlates poorly with affinity, a striking correlation is observed with molecular reach. Indeed, the molecular reach explains differences in neutralisation for antibodies binding with the same affinity to the same RBD-epitope. Thus, antibodies within an isotype class binding the same antigen can display differences in molecular reach, substantially modulating their binding and functional properties.
The cellular and molecular basis of defects in Down syndrome fetal bone marrow B-lymphopoiesis (DPhil thesis).
In this project, the identification and characterisation of new progenitor populations in FBM has facilitated existing models of fetal B-lymphopoiesis to be refined. In DS FBM, lymphopoiesis is disrupted with a severe block in B-cell differentiation that appears to occur upstream and is propagated through the newly described progenitors, as well as moderately impaired T-cell development and a skew toward NK lymphopoiesis. These findings were validated at the molecular level, with T21 causing broad changes in both the transcriptome and chromatin accessibility of FBM HSPCs.
Safety, tolerability, viral kinetics, and immune correlates of protection in healthy, seropositive UK adults inoculated with SARS-CoV-2: a single-centre, open-label, phase 1 controlled human infection study.
BACKGROUND: A SARS-CoV-2 controlled human infection model (CHIM) has been successfully established in seronegative individuals using a dose of 1×101 50% tissue culture infectious dose (TCID50) pre-alpha SARS-CoV-2 virus. Given the increasing prevalence of seropositivity to SARS-CoV-2, a CHIM that could be used for vaccine development will need to induce infection in those with pre-existing immunity. Our aim was to find a dose of pre-alpha SARS-CoV-2 virus that induced infection in previously infected individuals. METHODS: Healthy, UK volunteers aged 18-30 years, with proven (quantitative RT-PCR or lateral flow antigen test) previous SARS-CoV-2 infection (with or without vaccination) were inoculated intranasally in a stepwise dose escalation CHIM with either 1×101, 1×102, 1×10³, 1×104, or 1×105 TCID50 SARS-CoV-2/human/GBR/484861/2020, the same virus used in the seronegative CHIM. Post-inoculation, volunteers were quarantined in functionally negative pressure rooms (Oxford, UK) for 14 days and until 12-hourly combined oropharyngeal-nasal swabs were negative for viable virus by focus-forming assay. Outpatient follow-up continued for 12 months post-enrolment, with additional visits for those who developed community-acquired SARS-CoV-2 infection. The primary objective was to identify a safe, well tolerated dose that induced infection (defined as two consecutive SARS-CoV-2 positive PCRs starting 24 h after inoculation) in 50% of seropositive volunteers. This study is registered with ClinicalTrials.gov (NCT04864548); enrolment and follow-up to 12 months post-enrolment are complete. FINDINGS: Recruitment commenced on May 6, 2021, with the last volunteer enrolled into the dose escalation cohort on Nov 24, 2022. 36 volunteers were enrolled, with four to eight volunteers inoculated in each dosing group from 1×101 to 1×105 TCID50 SARS-CoV-2. All volunteers have completed quarantine, with follow-up to 12 months complete. Despite dose escalation to 1×105 TCID50, we were unable to induce sustained infection in any volunteers. Five (14%) of 36 volunteers were considered to have transient infection, based on the kinetic of their PCR-positive swabs. Transiently infected volunteers had significantly lower baseline mucosal and systemic SARS-CoV-2-specific antibody titres and significantly lower peripheral IFNγ responses against a CD8+ T-cell SARS-CoV-2 peptide pool than uninfected volunteers. 14 (39%) of 36 volunteers subsequently developed breakthrough infection with the omicron variant after discharge from quarantine. Most adverse events reported by volunteers in quarantine were mild, with fatigue (16 [44%]) and stuffy nose (16 [44%]) being the most common. There were no serious adverse events. INTERPRETATION: Our study demonstrates potent protective immunity induced by homologous vaccination and homologous or heterologous previous SARS-CoV-2 infection. The community breakthrough infections seen with the omicron variant supports the use of newer variants to establish a model with sufficient rate of infection for use in vaccine and therapeutic development. FUNDING: Wellcome Trust and Department for Health and Social Care.
Enhancer heterogeneity in acute lymphoblastic leukemia drives differential gene expression in patients.
Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. By generating high resolution Micro Capture-C data in primary patient samples, we identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may be a mechanism that drives transcriptional heterogeneity in cancer more generally.
Is noxious stimulus-evoked electroencephalography response a reliable, valid, and interpretable outcome measure to assess analgesic efficacy in neonates? A systematic review and individual participant data (IPD) meta-analysis protocol.
BACKGROUND: There are several major challenges limiting our ability to test analgesic efficacy for treatment of neonatal pain, and progress in analgesic drug studies in neonates has stalled. One significant issue is the reliance of clinical pain assessments on traditional behavioural and vital signs-based measures and the exclusion of novel brain-based biomarkers. In this review protocol, we outline our strategy to assess the reliability, validity, and interpretability of an electroencephalography (EEG)-based response biomarker for assessment of acute somatic nociceptive pain in neonates. METHODS: To standardise EEG analysis and generate the outcome of interest, we will perform an individual participant data (IPD) meta-analysis using data from neonates aged 34-44-week postmenstrual age that have had EEG recorded during acute somatic nociceptive skin-breaking procedures. Relevant data from both published and grey literature will be identified by searching six databases (MEDLINE, Embase, CINAHL, Web of Science, Scopus, Google Scholar), two clinical trial registry platforms (ClinicalTrials.gov, WHO ICTRP), and by consulting expert opinion. We will assess availability bias, data accuracy, and data quality by cross-referencing provided data with data descriptions in the literature, identifying duplicates and nonsensical values, and extracting quality control metrics. Data will be synthesised via a two-stage IPD meta-analysis using a random effects modelling approach grouped by site. Reliability (inter- and intra-rater) outcomes will be measured as Gwet's AC1 coefficient. Validity (known-groups and known-stimuli) outcomes will be measured as EEG response magnitude differences between clinically meaningfully different stimuli. Interpretability will be addressed by providing normative values, in both original and standardised units. DISCUSSION: The purpose of this study is to establish the reliability, validity, and interpretability of a specific EEG-based response biomarker for assessing acute somatic nociceptive pain in neonates. It will provide an overview of available data and how EEG is being used globally to assess acute neonatal pain. If sufficient IPD are made available and the outcome is reliable, valid, and interpretable, this work will support the use of EEG-based outcome measures as primary endpoints in clinical trials assessing analgesic efficacy in neonates. SYSTEMATIC REVIEW REGISTRATION: The protocol was registered with PROSPERO on 14 July 2023: CRD42023444809.
Platelets sequester extracellular DNA, capturing tumor-derived and free fetal DNA.
Platelets are anucleate blood cells vital for hemostasis and immunity. During cell death and aberrant mitosis, nucleated cells release DNA, resulting in "cell-free" DNA in plasma (cfDNA). An excess of cfDNA is deleterious. Given their ability to internalize pathogen-derived nucleic acids, we hypothesized that platelets may also clear endogenous cfDNA. We found that, despite lacking a nucleus, platelets contained a repertoire of DNA fragments mapping across the nuclear genome. We detected fetal DNA in maternal platelets and cancer-derived DNA in platelets from patients with premalignant and cancerous lesions. As current liquid biopsy approaches utilize platelet-depleted plasma, important genetic information contained within platelets is being missed. This study establishes a physiological role for platelets that has not previously been highlighted, with broad translational relevance.
AAV microdystrophin gene replacement therapy for Duchenne muscular dystrophy: progress and prospects.
Duchenne muscular dystrophy (DMD) is caused by pathogenic sequence variants occurring in the DMD gene which lead to the loss of the dystrophin protein, a molecular 'shock absorber' that protects muscle from contraction-induced injury. The large size of the dystrophin open reading frame precludes delivery of the full-length protein using a single adeno-associated virus (AAV) vector, which led to the development of internally-deleted dystrophin minigenes encoding partially-functional dystrophin. Indeed, five such microdystrophin therapies have been assessed in various clinical programmes. In 2023, Elevidys (Sarepta Therapeutics) received accelerated approval based on levels of dystrophin as a surrogate biomarker. In 2024, it received full approval despite unclear efficacy (i.e. not meeting primary or secondary outcomes in a phase 3 trial). Additionally, in 2025, two DMD individuals treated with Elevidys died after acute liver failure. A separate microdystrophin therapy, PF-06939926 (Pfizer) was discontinued for both efficacy and safety reasons (including the deaths of two clinical trial participants). Solid Biosciences, Genethon, REGENXBIO, and Insmed continue to develop microdystrophin therapies differing in transgene structure, promoter sequences, and AAV serotype. Here we describe recent progress in AAV-microdystrophin therapeutics development, and discuss the challenges facing such approaches, including pre-existing anti-capsid immunity, anti-transgene immunity, the unknown functionality of microdystrophin transgenes, transduction of muscle stem cells, and long-term transgene persistence.