Clarissa Prazeres da Costa

The global burden of infectious disease is on the rise, driven by antibiotic resistance, emerging pathogens, and gaps in accessible, decentralized diagnostics. To address these demands, there is a need for the development of diagnostic platforms for detection, surveillance, or monitoring assays with quick turnaround times. Oxford Nanopore sequencing, a platform for real-time long-read third-generation sequencing, has emerged as a promising tool in this space, with the ability to detect a wide range of molecular targets, including DNA, RNA, proteins, and base modifications. Its unique advantages, including portability, scalability, and the capacity for culture-independent workflows, combined with the depth of sequencing data produced, make it especially well-suited for infectious disease diagnostics and surveillance in both clinical and field settings. In this review, we aim to critically examine recent advances in the application of nanopore sequencing for diagnosing infectious diseases caused by bacterial, viral, fungal, and parasitic agents, as well as for detecting antimicrobial resistance genes. We also compare its potential advantages over conventional diagnostic methods and explore its expanding role in tracking outbreaks, monitoring pathogens in the environment, and detecting spillover events from animals to humans. Together, these applications highlight the relevance and position of this novel technology as a tool of choice for translational medical research and clinicians in the fight against infectious diseases and for global health preparedness.

Schistosomiasis, a neglected tropical disease caused by infection with parasitic blood flukes of the genus Schistosoma , affects more than 250 million people worldwide. Currently, patients rely on a single medicine, praziquantel (PZQ), discovered half a century ago. To combat drug resistance and reach elimination of the schistosome parasite, alternative drugs to PZQ are required that treat and prevent disease. Here, we describe a drug candidate, M5493, with potent activity against multiple parasite life cycle stages. The target is a parasite transient receptor potential ion channel, distinct from the target of PZQ. To circumvent its limited bioavailability, the active agent was converted to prodrug M5493, which displayed robust activity against the schistosome parasite at doses as low as 5 milligrams per kilogram in mice infected with Schistosoma mansoni . Unlike the benzodiazepine meclonazepam, which displays clinical antihelminthic activity through the same target, M5493 showed no sedating side effects given that it did not engage GABA _A receptors. Overall, this compound has the potential to provide single-dose treatment, broad-spectrum efficacy against schistosome parasites at different life cycle stages, and transmission blocking activity.

Objectives: Helminth parasites infect over a quarter of the global population and can profoundly modulate host immunity, potentially influencing vaccine performance and the spread of pandemic pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite the high global endemicity of helminth infections, their impact on immune responses to various COVID-19 vaccines remains unknown. This study aimed to evaluate the impact of Schistosoma infection on the immunogenicity and protective efficacy of messenger RNA (mRNA)- and protein-based COVID-19 vaccines. Methods: Mice with Schistosoma infection and non-infected controls were immunized with either an mRNA-based COVID-19 vaccine or an alum-adjuvanted spike protein vaccine. Vaccine-induced humoral and cellular immune responses were assessed, and protective efficacy was evaluated using a SARS-CoV-2 challenge model. Results: COVID-19 mRNA vaccination induced strong spike-specific antibody and CD4 T-cell responses in Schistosoma -infected mice comparable to non-infected controls, despite a Th2/regulatory-biased immune environment, although multifunctional CD8 T-cell responses were reduced. Alum-adjuvanted protein vaccination elicited robust humoral but weaker cellular immunity, with comparable immune responses in infected and non-infected mice. Following SARS-CoV-2 challenge, both vaccine platforms conferred effective protection, with substantial viral clearance and minimal lung pathology. Conclusions: mRNA and protein vaccines elicit distinct immune profiles; however, both protect effectively against SARS-CoV-2 infection in mice with concurrent helminth infection.

Background: Schistosomiasis, one of the most prevalent neglected tropical diseases, is treated exclusively with praziquantel, which targets only adult worms. High reinfection rates and the potential emergence of drug resistance emphasize the need for a deeper understanding of host-parasite developmental biology for innovative therapeutic targets. Most studies have focused on parasite-derived immunomodulators and proteolytic enzymes essential for host invasion, while the contribution of host-derived factors to parasite development has remained largely unexplored. Here, we identify and functionally characterize a host phospholipase that modulates parasite diacylglycerophospholipid metabolism and development. Methods: Large-scale proteomic screening identified host platelet-activating factor acetylhydrolase (PAFAH) as a candidate schistosomicidal factor. Using a mouse model of schistosomiasis, we produced recombinant mouse PAFAH (MsPAFAH) and performed ex vivo killing assays on juvenile stages and adult worms. Parasite morphology and reproductive organ integrity were assessed by confocal and scanning electron microscopy. Alterations in glycerophospholipid distribution were quantified by UPLC-MS/MS-based fatty acid profiling, followed by rescue experiments with free fatty acid supplementation. Results: MsPAFAH was upregulated in schistosome-infected mice and exhibited potent schistosomicidal activity against all life stages in the host. MsPAFAH treatment led to profound impairments in worm fecundity, pairing stability, reproductive organ integrity, and stem cell development. This activity was associated with substantial sex-dependent changes in ether-phospholipid composition and distribution within the schistosome. MsPAFAH specifically decreased the availability of phospholipid species containing namely eicosenoic (20:1) and docosatetraenoic acid (22:4), while increasing levels of respective hydrolysis (lyso) products carrying 20:1 or stearic acid (18:0), predominantly in males. Supplementation of metabolized fatty acids C20:1/eicosenoic acid and 22:4/adrenic acid rescued the viability of female worms, confirming the essential role of the metabolism of these diacylglycerophospholipids in schistosome survival. Conclusions: Our findings reveal a previously unrecognized host-derived regulator of schistosome development, highlighting phospholipid metabolism as a promising therapeutic axis for consideration in schistosomiasis interventions.

Background: Effective diagnosis of Schistosoma haematobium is critical for disease management. However, current tools often lack sensitivity for low-intensity infections, a challenge noted in the recent WHO Roadmap for Neglected Tropical Diseases. This study aimed to evaluate a novel cell-free DNA (cfDNA) quantitative Polymerase Chain Reaction (qPCR) assay, utilizing 20 µl of plasma with crude DNA extraction. This approach intends to improve diagnostic accuracy and lower implementation barriers for molecular tests in field settings. We compared its performance against urine filtration microscopy and the Up-converting Reporter Particle Lateral Flow Circulating Anodic Antigen (UCP-LF CAA) test in pregnant women from Lambaréné, Gabon. Methods: A prospective cross-sectional study was conducted on 296 pregnant women in Gabon. EDTA blood, urine, and stool samples were collected from December 2018 to November 2020. Urine samples were analyzed by urine filtration microscopy and UCP-LF CAA. The cfDNA qPCR assay was performed retrospectively on 20 µl of frozen plasma using a simplified DNA extraction method. Diagnostic accuracy was assessed via sensitivity, specificity, and agreement measures. Bayesian latent class analysis (BLCA) was used to estimate test performance in the absence of a gold standard. Results: Diagnostic positivity varied: 24.3% by cfDNA qPCR, 18.6% by urine filtration microscopy, and 20.9% by UCP-LF CAA. Agreement between tests was limited. BLCA indicated qPCR to be the most sensitive (74.0%, 95% CrI: 57.2–90.8), followed by UCP-LF CAA (65.7%, 95% CrI: 49.0–82.8). Microscopy was the most specific (94.9%, 95% CrI : 89.6–99.3). Estimated prevalence ranged between 22.5 and 27.1%. Receiver operating characteristic (ROC) analysis confirmed good individual performance (AUC 0.837–0.868), with performance improving when combining any two tests (AUC up to 0.931). Conclusions: This study validates the high sensitivity of a novel cfDNA qPCR approach using only 20 µl of plasma, demonstrating performance comparable to microscopy and UCP-LF CAA. While microscopy maintains high specificity, combining it with a high-sensitivity test such as qPCR or UCP-LF CAA provides significant diagnostic improvement. Further optimizing the specificity of this streamlined qPCR assay brings us closer to a highly accurate, feasible point-of-care diagnostic crucial for schistosomiasis control programs in resource-limited settings.

Maternal immune activation (MIA) refers to an immune response triggered in a pregnant mother by infections, inflammation, or other immune challenges that can impact offspring health. We propose aligning MIA within the framework of the developmental origins of health and disease (DOHaD) theory because it has the potential to provide mechanistic evidence for long‐term outcomes of fetomaternal crosstalk disruptions. MIA models are created by exposing pregnant animals to immune‐activating agents such as inosinic–polycytidylic acid (poly I:C) or lipopolysaccharide (LPS), which mimic viral or bacterial infections, respectively. Next to these acute MIA models, chronic helminth infections during pregnancy have been employed as an additional, more physiological model of infection. MIA models have helped researchers explore how maternal infections during pregnancy may impact the offspring's risk of neurodevelopmental disorders. Emerging evidence suggests that these models have a broader impact on organ development, the immune system, and, consequently, immune‐related disorders such as allergies. Our review focuses on evidence derived mainly from mouse models of MIA that have investigated maternal signals, such as cytokines and microbiota, on fetal hematopoiesis, adults’ immune cell compartments, including the bone marrow, and their relation to the development of offspring allergies. Where applicable, studies from other species are indicated.

Background: Digital platforms play a vital role in improving the availability and access to health research outputs, enhancing the engagement of policy makers and practitioners in the research processes. Despite their potential, it needs to be explored how digital platforms are used to manage and share health research datasets and publications, and to translate research findings among health networks or institutions in sub-Saharan Africa (SSA). Objective: This study aimed to assess the practices of health research data management, including sharing among researchers and their support staff within 3 large research networks for health innovations in SSA. Methods: A cross-sectional mixed methods survey was conducted across 3 research networks in SSA, showing experiences of sharing research data using digital platforms among researchers of 3 large research and innovation networks in SSA and affiliated institutions in the Global North. A total of 160 respondents completed a self-administered web-based questionnaire, and following data cleansing, the survey data were analyzed using both descriptive and inferential statistics. Results: Most respondents (91/160, 56.9%) used electronic data collection tools to collect research data. Almost half (79/160, 49.4%) of the respondents have a digital research data management platform. More than half of the respondents shared their research datasets (102/160, 63.8%), and 61.3% (98/160) shared research findings with the research community through different channels. Furthermore, most respondents shared their research datasets and research outputs through institutional data repositories (42/160, 26.1%), scientific conferences (123/160, 76.9%), and journal articles (110/160, 68.8%). This study found that parameters such as sex, professional category (health professional, information and communication technology professional, and data managers), and the role (researcher or student) influence health research data sharing within the community. The results show that the roles of the individual have the strongest association with the sharing of research datasets, followed by years of experience in research, then sex, and profession. Females were less likely to share their research datasets than males. Data managers and information and communication technology professionals exchanged datasets less frequently in the professional group, and the researcher’s role was statistically significant in sharing research datasets. Conclusions: This study demonstrates that most researchers share research datasets and outputs through various channels. It was further found that digital platforms were essential in managing and sharing research datasets and publications since more than half (85/160, 53.1%) of the respondents have and use digital platforms. In addition, the study identified factors that influenced researchers’ practices of sharing research datasets and publications. Furthermore, key gaps limit the sharing of these research datasets, including inadequate infrastructure, insufficient African dataset sharing platforms, a lack of institutional policy, and limited skills to use available platforms.

In this chapter we explore the intricate relationship between schistosomes and their human hosts, delving into the biology, pathogenesis, clinical manifestations, and diagnostics of schistosomiasis. Prevalent for millennia, the disease remains a significant global health challenge, particularly in sub-Saharan Africa. The life cycle of schistosomes involves freshwater snails, with transmission to the definitive host, such as humans, linked to water exposure. Acute and chronic stages manifest with diverse symptoms, ranging from fever and hematuria to severe organ damage, including fibrosis and bladder cancer. Advanced diagnostics, such as molecular and serological methods, complement traditional microscopy but are less accessible in endemic regions. Clinical interventions focus primarily on praziquantel, with significant reversibility of symptoms even in late stages. The chapter underscores schistosomiasis as a “One Health” issue, requiring integrated approaches targeting humans, snails, and environmental water sources. It highlights the disease’s socioeconomic impact and its interplay with other infections, including HIV. Comprehensive elimination strategies encompassing diagnostics, treatment, and environmental control are emphasized to combat this ancient yet persistent parasitic challenge.

Introduction: In human neurocysticercosis (NCC), the cellular and molecular mechanisms of host-parasite interactions triggering brain inflammation and epileptic seizures in Sub-Saharan Africa are poorly understood. Emerging evidence indicates that the viability of the cyst of the pork tapeworm Taenia solium determines brain inflammation and, thus, symptom development and disease severity. We have previously shown that while viable cyst-released molecules promote immune regulation and often asymptomatic disease, the fluid from degenerating cysts causes inflammation in microglia and peripheral immune cells, potentially driving immune-mediated pathology. This study aims to elucidate the apoptotic signaling pathways underlying this process and their relevance for symptomatic disease in NCC patients.Materials and methods: Human and porcine peripheral immune cells, as well as murine microglia, were exposed to T. solium cyst vesicular fluid (CVF). Apoptosis signaling pathways were analysed using flow cytometric FLICA (fluorochrome-labeled inhibitors of caspases) caspase 8 and 9 assays, while mitochondrial dysfunction was assessed via TMRE and MitoTracker Deep Red and Green fluorescent probes. Apoptosis-inducing CVF molecules were identified by differential mass spectrometry and functionally tested using specific inhibitors. Caspase activity and soluble mediators (FasL, ROS, TNFα) were measured in NCC asymptomatic and symptomatic patients’ sera, and inflammatory T cell infiltrates expressing caspases near viable and degenerating cysts in naturally infected pig brain slices were examined via immunohistology.Results: We found that vesicular fluid derived from cysts primarily induced apoptosis and caspase 3 and 9 activity, and only minimal necrosis, in a dose-dependent manner across central and peripheral immune cells. This effect was prominent in CD16⁺ monocytes, microglia, and in CD3⁺ T cell-expressing caspase 3 near degenerating brain cysts. Apoptotic signaling was predominantly mediated by a dynamic remodeling of caspase 9 pathway, accompanied by a significant loss of mitochondrial potential and a sharp decrease in Bid and Bcl2 transcription, favoring the intrinsic over the FasL-dependent extrinsic pathway and mechanisms. This process is primarily mediated by small molecules (< 30 kDa), and remained unaffected by heat and proteinase treatment. Notably, symptomatic NCC patients exhibited elevated FasL levels correlating with increased caspase activity, underscoring the potential contribution of apoptosis to disease pathogenesis.Conclusions: This study identifies caspase 9-mediated apoptosis as a mechanism of helminth-induced brain inflammation and implicates FasL in symptomatic disease progression. These insights enhance our understanding of NCC immunopathogenesis and may inform future therapeutic strategies targeting apoptotic pathways.

Background: Neurocysticercosis (NCC) and HIV co-infection frequently occur in sub-Saharan Africa, yet the accuracy of available serological tests for NCC in immunosuppressed patients is uncertain. Methodology: We performed a cross-sectional diagnostic study on 101 people living with HIV from two endemic districts in Tanzania. Participants provided serum for cysticercosis antigen ELISA and Western Blot IgG; any positive result prompted neuroimaging investigation with cerebral computed tomography. NCC was diagnosed according to the 2017 revised Del Brutto criteria based on cCT according to Del Brutto criteria modified to exclude serology. Sensitivity, specificity, and area under the receiver–operating–characteristic curve (AUC) were calculated and adjusted for CD4 count and HIV stage. Two algorithms were compared: parallel testing (“either-test-positive”) and sequential screening (Ag ELISA screen, western blot IgG confirm). Results: NCC prevalence was 23%. Western Blot IgG outperformed Ag ELISA (sensitivity 57% vs. 30%; specificity 87% vs. 86%; AUC 0.73 vs. 0.57). Western blot IgG sensitivity declined to 54% when CD4 < 500 cells µL⁻¹, while Ag ELISA remained low. Western blot IgG positivity independently predicted NCC (adjusted odds ratio 4.1, 95% CI 1.4–11.9); Ag ELISA did not. When we counted a positive if either test was positive (parallel rule), sensitivity rose to 78% and NPV to 87%. When we ran Ag ELISA only if IgG was negative (sequential rule), we saved 70% of IgG strips, kept specificity at 95%, and PPV at 69%, but sensitivity fell to 39%. Conclusions: Western blot IgG is the most reliable single serological test for NCC in PLHIV. Parallel testing increased sensitivity and NPV and may suit better primary-level facilities without routine imaging. Sequential testing achieved high specificity, PPV, and conserved test kits, making it ideal for centers with limited reagents or scanner access. Tiered use of these assays can streamline NCC diagnosis in T. solium endemic, resource-limited settings.