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Abstract Background Lymphatic filariasis (LF) is a globally significant, vector-borne, neglected tropical disease that can result in severe morbidity and disability. As the World Health Organization (WHO) Global Programme to Eliminate Lymphatic Filariasis makes progress towards LF elimination, there is greater need to develop sensitive strategies for post-intervention surveillance. Molecular xenomonitoring (MX), the detection of pathogen DNA in vectors, may provide a sensitive complement to traditional human-based surveillance techniques, including detection of circulating filarial antigen and microfilaraemia (Mf). This study aims to explore the relationship between human Mf prevalence and the prevalence of polymerase chain reaction (PCR)-positive mosquitoes using MX. Methods This study compared Mf and MX results from a 2019 community-based survey conducted in 35 primary sampling units (PSUs) in Samoa. This study also investigated concordance between presence and absence of PCR-positive mosquitoes and Mf-positive participants at the PSU level, and calculated sensitivity and negative predictive values for each indicator using presence of any Mf-positive infection in humans or PCR-positive mosquitoes as a reference. Correlation between prevalence of filarial DNA in mosquitoes and Mf in humans was estimated at the PSU and household/trap level using mixed-effect Bayesian multilevel regression analysis. Results Mf-positive individuals were identified in less than half of PSUs in which PCR-positive mosquito pools were present (13 of 28 PSUs). Prevalence of PCR-positive mosquitoes (each species separately) was positively correlated with Mf prevalence in humans at the PSU level. Analysed at the species level, only Aedes polynesiensis demonstrated strong evidence of positive correlation (r) with human Mf prevalence at both PSU (r: 0.5, 95% CrI 0.1–0.8) and trap/household levels (r: 0.6, 95% CrI 0.2–0.9). Conclusions Findings from this study demonstrate that MX can be a sensitive surveillance method for identifying residual infection in low Mf prevalence settings. MX identified more locations with signals of transmission than Mf-testing. Strong correlation between estimated PCR-positive mosquitoes in the primary vector species and Mf in humans at small spatial scales demonstrates the utility of MX as an indicator for LF prevalence in Samoa and similar settings. Further investigation is needed to develop MX guidelines to strengthen the ability of MX to inform operational decisions. Graphical abstract

Abstract Background Soil-transmitted helminths infect an estimated 18% of the world’s population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets. Methods We aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences. Results For Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28–0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement. Conclusions There is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR. Graphical abstract

Parafilaroides decorus, also known as sea lion lungworm, is a metastrongyloid nematode that infects otariid hosts, such as the charismatic California sea lion, Zalophus californianus. P. decorus causes bronchointerstitial pneumonia, respiratory distress, reduced ability to swim, dive and hunt and as a result, increased mortality particularly in young animals. Respiratory disease is a leading cause of stranding and admission to rehabilitation centers on the Pacific coast. Low-coverage genomic sequencing of four P. decorus individuals analyzed through Galaxy's RepeatExplorer identified a novel repeat DNA family we employed to design a sensitive quantitative PCR (qPCR) assay for diagnosing infections from fecal or sputum samples. The assay detects as little as 10 fg of P. decorus DNA and a linear regression model developed using a standard curve can be used to estimate the concentration of P. decorus DNA in a sample, ± 0.015 ng. This knowledge can be leveraged to estimate the level of parasite burden, which can be used to design improved treatments for animals in rehabilitation. Improved treatment of infections will aid in more animals being successfully released back into the wild.

Abstract Background Japan is one of the few countries believed to have eliminated soil-transmitted helminths (STHs). In 1949, the national prevalence of Ascaris lumbricoides was 62.9%, which decreased to 0.6% in 1973 due to improvements in infrastructure, socioeconomic status, and the implementation of national STH control measures. The Parasitosis Prevention Law ended in 1994 and population-level screening ceased in Japan; therefore, current transmission status of STH in Japan is not well characterized. Sporadic cases of STH infections continue to be reported, raising the possibility of a larger-scale recrudescence of STH infections. Given that traditional microscopic detection methods are not sensitive to low-intensity STH infections, we conducted targeted prevalence surveys using sensitive PCR-based assays to evaluate the current STH-transmission status and to describe epidemiological characteristics of areas of Japan believed to have achieved historical elimination of STHs. Methods Stool samples were collected from 682 preschool- and school-aged children from six localities of Japan with previously high prevalence of STH. Caregivers of participants completed a questionnaire to ascertain access to water, sanitation and hygiene (WASH), and potential exposures to environmental contamination. For fecal testing, multi-parallel real-time PCR assays were used to detect infections of Ascaris lumbricoides, Necator americanus, Ancylostoma duodenale and Trichuris trichiura. Results Among the 682 children, no positive samples were identified, and participants reported high standards of WASH. Conclusions To our knowledge, this is the first STH-surveillance study in Japan to use sensitive molecular techniques for STH detection. The results suggest that recrudescence of STH infections has not occurred, and that declines in prevalence have been sustained in the sampled areas. These findings suggest that reductions in prevalence below the elimination thresholds, suggestive of transmission interruption, are possible. Additionally, this study provides circumstantial evidence that multi-parallel real-time PCR methods are applicable for evaluating elimination status in areas where STH prevalence is extremely low.

Background Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making. Methodology/Principal findings Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (SchistosomajaponicumTandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum. Conclusions/Significance The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts. Author summary Schistosomiasis is a Neglected Tropical Disease (NTD) estimated to infect more than 230 million people worldwide. Of the various species of schistosomes that cause disease in humans, Schistosoma japonicum is considered the most virulent. As such, this pathogen presents a crucial public health threat. Typically, diagnosis of S. japonicum has been performed via the Kato-Katz technique which has been shown to dramatically underestimate the burden of infection, resulting in a need for improved detection strategies. To address this need, we have employed bioinformatic tools in order to identify a tandemly arranged, highly repetitive, DNA sequence, SjTR1, in the S. japonicum genome. Utilizing this sequence as a real-time PCR assay target, we have developed a sensitive and specific assay for the detection of S. japonicum DNA. Employment of this assay in field settings will facilitate the accurate detection of S. japonicum and provide guidance capable of informing mass drug administration efforts targeting elimination.

Molecular xenomonitoring (MX), the detection of filarial DNA in mosquitoes using molecular methods (PCR), is a potentially useful surveillance strategy for lymphatic filariasis (LF) elimination programs. Delay in filarial antigen (Ag) clearance post-treatment is a limitation of using human surveys to provide an early indicator of the impact of mass drug administration (MDA), and MX may be more useful in this setting. We compared prevalence of infected mosquitoes pre- and post-MDA (2018 and 2019) in 35 primary sampling units (PSUs) in Samoa, and investigated associations between the presence of PCR-positive mosquitoes and Ag-positive humans. We observed a statistically significant decline in estimated mosquito infection prevalence post-MDA at the national level (from 0.9% to 0.3%, OR 0.4) but no change in human Ag prevalence during this time. Ag prevalence in 2019 was higher in randomly selected PSUs where PCR-positive pools were detected (1.4% in ages 5–9; 4.8% in ages ≥10), compared to those where PCR-positive pools were not detected (0.2% in ages 5–9; 3.2% in ages ≥10). Our study provides promising evidence for MX as a complement to human surveys in post-MDA surveillance.

Abstract Background The strategy of pooling stool specimens has been extensively used in the field of parasitology in order to facilitate the screening of large numbers of samples whilst minimizing the prohibitive cost of single sample analysis. The aim of this study was to develop a standardized reproducible pooling protocol for stool samples, validated between two different laboratories, without jeopardizing the sensitivity of the quantitative polymerase chain reaction (qPCR) assays employed for the detection of soil-transmitted helminths (STHs). Two distinct experimental phases were recruited. First, the sensitivity and specificity of the established protocol was assessed by real-time PCR for each one of the STHs. Secondly, agreement and reproducibility of the protocol between the two different laboratories were tested. The need for multiple stool sampling to avoid false negative results was also assessed. Finally, a cost exercise was conducted which included labour cost in low- and high-wage settings, consumable cost, prevalence of a single STH species, and a simple distribution pattern of the positive samples in pools to estimate time and money savings suggested by the strategy. Results The sensitivity of the pooling method was variable among the STH species but consistent between the two laboratories. Estimates of specificity indicate a ‘pooling approach’ can yield a low frequency of ‘missed’ infections. There were no significant differences regarding the execution of the protocol and the subsequent STH detection between the two laboratories, which suggests in most cases the protocol is reproducible by adequately trained staff. Finally, given the high degree of agreement, there appears to be little or no need for multiple sampling of either individuals or pools. Conclusions Our results suggest that the pooling protocol developed herein is a robust and efficient strategy for the detection of STHs in ‘pools-of-five’. There is notable complexity of the pool preparation to ensure even distribution of helminth DNA throughout. Therefore, at a given setting, cost of labour among other logistical and epidemiological factors, is the more concerning and determining factor when choosing pooling strategies, rather than losing sensitivity and/or specificity of the molecular assay or the method.

Eukaryotic parasites are significant contributors to childhood illness in Niger. While helminthiases have received national attention through mass deworming efforts, the epidemiology of intestinal protozoa in Niger remains underexamined. This study employed real-time PCR diagnostics to describe the prevalence of two schistosomes, four soil-transmitted helminths, and one protozoan parasite in Boboye Department, Dosso Region. Prevalence was assessed using bulk stool specimens collected from a population-based sample of 86 children residing in 9 communities. Anthropometric measurements were used to calculate child growth z-scores and stool consistency was graded. Helminths were absent from the study population, with the exception of a single Schistosoma haematobium infection (1/86; 1.2%). Giardia duodenalis was the only protozoa present, detected in 65% (56/86) of children. Prevalence of G. duodenalis peaked in 2-year-olds with 88% (15/17) positivity. The population was generally undernourished, though growth indices did not differ significantly between children with and without G. duodenalis infection.

Background: Results from an increasing number of studies suggest that mosquito excreta/feces (E/F) testing has considerable potential to serve as a supplement for traditional molecular xenomonitoring techniques. However, as the catalogue of possible use-cases for this methodology expands, and the list of amenable pathogens grows, a number of fundamental methods-based questions remain. Answering these questions is critical to maximizing the utility of this approach and to facilitating its successful implementation as an effective tool for molecular xenomonitoring. Methods: Utilizing E/F produced by mosquitoes or tsetse flies experimentally exposed to Brugia malayi, Plasmodium falciparum, or Trypanosoma brucei brucei, factors such as limits of detection, throughput of testing, adaptability to use with competent and incompetent vector species, and effects of additional blood feedings post parasite-exposure were evaluated. Two platforms for the detection of pathogen signal (quantitative real-time PCR and digital PCR (dPCR)) were also compared, with strengths and weaknesses examined for each. Results: Experimental results indicated that high throughput testing is possible when evaluating mosquito E/F for the presence of either B. malayi or P. falciparum from both competent and incompetent vector mosquito species. Furthermore, following exposure to pathogen, providing mosquitoes with a second, uninfected bloodmeal did not expand the temporal window for E/F collection during which pathogen detection was possible. However, this collection window did appear longer in E/F collected from tsetse flies following exposure to T. b. brucei. Testing also suggested that dPCR may facilitate detection through its increased sensitivity. Unfortunately, logistical obstacles will likely make the large-scale use of dPCR impractical for this purpose. Conclusions: By examining many E/F testing variables, expansion of this technology to a field-ready platform has become increasingly feasible. However, translation of this methodology from the lab to the field will first require field-based pilot studies aimed at assessing the efficacy of E/F screening.

During 2012–2015, US-bound refugees living in Myanmar–Thailand border camps (n = 1,839) were surveyed for hookworm infection and treatment response by using quantitative PCR. Samples were collected at 3 time points: after each of 2 treatments with albendazole and after resettlement in the United States. Baseline prevalence of Necator americanus hookworm was 25.4%, Ancylostoma duodenale 0%, and Ancylostoma ceylanicum (a zoonosis) 5.4%. Compared with N. americanus prevalence, A. ceylanicum hookworm prevalence peaked in younger age groups, and blood eosinophil concentrations during A. ceylanicum infection were higher than those for N. americanus infection. Female sex was associated with a lower risk for either hookworm infection. Cure rates after 1 dose of albendazole were greater for A. ceylanicum (93.3%) than N. americanus (65.9%) hookworm (p

The distinct evolutionary pressures faced by Pinnipeds have likely resulted in strong coevolutionary ties to their parasites (Leidenberger et al., 2007). This study focuses on the phocid seal filarial heartworm species Acanthocheilonema spirocauda. A. spirocauda is known to infect a variety of phocid seals, but does not appear to be restricted to a single host species (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). However, to date, seal heartworm has never been reported in grey seals (Halichoerus grypus) (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). The proposed vector for seal heartworm is Echinophthirius horridus, the seal louse. Seal lice are known to parasitize a wide array of phocid seal species, including the grey seal. With the advent of climate change, disease burden is expected to increase across terrestrial and marine mammals (Harvell et al., 2002). Accordingly, increased prevalence of seal heartworm has recently been reported in harbor seals (Phoca vitulina) (Lehnert et al., 2015). Thus, the need for improved, rapid, and cost-effective diagnostics is urgent. Here we present the first A. spirocauda-specific rapid diagnostic test (a quantitative real-time PCR assay), based on a highly repetitive genomic DNA repeat identified using whole genome sequencing and subsequent bioinformatic analysis. The presence of an insect vector provides the opportunity to develop a multifunctional diagnostic tool that can be used not only to detect the parasite directly from blood or tissue specimens, but also as a molecular xenomonitoring (XM) tool that can be used to assess the epidemiological profile of the parasite by screening the arthropod vector. Using this assay, we provide evidence for the first reported case of seal heartworm in a grey seal. Keywords: Harbor seal, Parasites, Seal heartworm, Acanthocheilonema spirocauda, Real-time PCR, Molecular diagnostics, Xenomonitoring, Genomic repeats

The lemurs of Madagascar are threatened by human activities. We present the first molecular detection of canine heartworm (Dirofilaria immitis) in a wild non-human primate, the mouse lemur (Microcebus rufus). Zoonotic D. immitis infection has been associated with clinical pathology that includes serious and often fatal cardiac and pulmonary reactions. With human encroachment and associated increases in free-roaming dog populations in Madagascar, we examined lemurs for zoonotic canid pathogens. D. immitis presents a new potential conservation threat to lemurs. We highlight the need for wide-ranging and effective interventions, particularly near protected areas, to address this growing conservation issue. Keywords: Madagascar conservation, Pathogen spillover, Mosquito-borne disease, Blood parasite, Canine vector-borne diseases, brown mouse lemur, Microcebus rufus

The use of microfilaricidal drugs for the control of onchocerciasis and lymphatic filariasis (LF) necessitates prolonged yearly dosing. Prospects for elimination or eradication of these diseases would be enhanced by the availability of a macrofilaricidal drug. Flubendazole (FLBZ), a benzimidazole anthelmintic, is an appealing candidate. FLBZ has demonstrated potent macrofilaricidal effects in a number of experimental rodent models and in one human trial. Unfortunately, FLBZ was deemed unsatisfactory for use in mass drug administration campaigns due to its limited oral bioavailability. A new formulation that enables sufficient bioavailability following oral administration could render FLBZ an effective treatment for onchocerciasis and LF. Identification of drug-derived effects is important in ascertaining a dosage regimen which is predicted to be lethal to the parasite in situ. In previous histological studies, exposure to FLBZ induced damage to tissues required for reproduction and survival at pharmacologically relevant concentrations. However, more precise and quantitative indices of drug effects are needed. This study assessed drug effects using a transcriptomic approach to confirm effects observed histologically and to identify genes which were differentially expressed in treated adult female Brugia malayi. Comparative analysis across different concentrations (1 μM and 5 μM) and durations (48 and 120 h) provided an overview of the processes which are affected by FLBZ exposure. Genes with dysregulated expression were consistent with the reproductive effects observed via histology in our previous studies. This study revealed transcriptional changes in genes involved in embryo development. Additionally, significant downregulation was observed in genes encoding cuticle components, which may reflect changes in developing embryos, the adult worm cuticle or both. These data support the hypothesis that FLBZ acts predominantly on rapidly dividing cells, and provides a basis for selecting molecular markers of drug-induced damage which may be of use in predicting efficacious FLBZ regimens.

The balance of expense and ease of use vs. specificity and sensitivity in diagnostic assays for helminth disease is an important consideration, with expense and ease often winning out in endemic areas where funds and sophisticated equipment may be scarce. In this review, we argue that molecular diagnostics, specifically new assays that have been developed with the aid of next-generation sequence data and robust bioinformatic tools, more than make up for their expense with the benefit of a clear and precise assessment of the situation on the ground. Elimination efforts associated with the London Declaration and the World Health Organization (WHO) 2020 Roadmap have resulted in areas of low disease incidence and reduced infection burdens. An accurate assessment of infection levels is critical for determining where and when the programs can be successfully ended. Thus, more sensitive assays are needed in locations where elimination efforts are approaching a successful conclusion. Although microscopy or more general PCR targets have a role to play, they can mislead and cause study results to be confounded. Hyper-specific qPCR assays enable a more definitive assessment of the situation in the field, as well as of shifting dynamics and emerging diseases.

Background: Molecular xenomonitoring (MX), the testing of insect vectors for the presence of human pathogens, has the potential to provide a non-invasive and cost-effective method for monitoring the prevalence of disease within a community. Current MX methods require the capture and processing of large numbers of mosquitoes, particularly in areas of low endemicity, increasing the time, cost and labour required. Screening the excreta/feces (E/F) released from mosquitoes, rather than whole carcasses, improves the throughput by removing the need to discriminate vector species since non-vectors release ingested pathogens in E/F. It also enables larger numbers of mosquitoes to be processed per pool. However, this new screening approach requires a method of efficiently collecting E/F. Methods: We developed a cone with a superhydrophobic surface to allow for the efficient collection of E/F. Using mosquitoes exposed to either Plasmodium falciparum, Brugia malayi or Trypanosoma brucei brucei, we tested the performance of the superhydrophobic cone alongside two other collection methods. Results: All collection methods enabled the detection of DNA from the three parasites. Using the superhydrophobic cone to deposit E/F into a small tube provided the highest number of positive samples (16 out of 18) and facilitated detection of parasite DNA in E/F from individual mosquitoes. Further tests showed that following a simple washing step, the cone can be reused multiple times, further improving its cost-effectiveness. Conclusions: Incorporating the superhydrophobic cone into mosquito traps or holding containers could provide a simple and efficient method for collecting E/F. Where this is not possible, swabbing the container or using the washing method facilitates the detection of the three parasites used in this study.

Background: Molecular xenomonitoring (MX), the testing of insect vectors for the presence of human pathogens, has the potential to provide a non-invasive and cost-effective method for monitoring the prevalence of disease within a community. Current MX methods require the capture and processing of large numbers of mosquitoes, particularly in areas of low endemicity, increasing the time, cost and labour required. Screening the excreta/feces (E/F) released from mosquitoes, rather than whole carcasses, improves the throughput by removing the need to discriminate vector species since non-vectors release ingested pathogens in E/F. It also enables larger numbers of mosquitoes to be processed per pool. However, this new screening approach requires a method of efficiently collecting E/F. Methods: We developed a cone with a superhydrophobic surface to allow for the efficient collection of E/F. Using mosquitoes exposed to either Plasmodium falciparum, Brugia malayi or Trypanosoma brucei brucei, we tested the performance of the superhydrophobic cone alongside two other collection methods. Results: All collection methods enabled the detection of DNA from the three parasites. Using the superhydrophobic cone to deposit E/F into a small tube provided the highest number of positive samples (16 out of 18) and facilitated detection of parasite DNA in E/F from individual mosquitoes. Further tests showed that following a simple washing step, the cone can be reused multiple times, further improving its cost-effectiveness. Conclusions: Incorporating the superhydrophobic cone into mosquito traps or holding containers could provide a simple and efficient method for collecting E/F. Where this is not possible, swabbing the container or using the washing method facilitates the detection of the three parasites used in this study.

Background Embolic stroke of unknown source (ESUS) accounts for 1 in 6 ischemic strokes. Current guidelines do not recommend routine cardiac magnetic resonance (CMR) imaging in ESUS, and beyond the identification of cardioembolic sources, there are no data assessing new clinical findings from CMR in ESUS. This study aimed to assess the prevalence of new cardiac and noncardiac findings and to determine their impact on clinical care in patients with ESUS. Methods and Results In this prospective, multicenter, observational study, CMR imaging was performed within 3 months of ESUS. All scans were reported according to standard clinical practice. A new clinical finding was defined as one not previously identified through prior clinical evaluation. A clinically significant finding was defined as one resulting in further investigation, follow‐up, or treatment. A change in patient care was defined as initiation of medical, interventional, surgical, or palliative care. From 102 patients recruited, 96 underwent CMR imaging. One or more new clinical findings were observed in 59 patients (61%). New findings were clinically significant in 48 (81%) of these patients. Of 40 patients with a new clinically significant cardiac finding, 21 (53%) experienced a change in care (medical therapy, n=15; interventional/surgical procedure, n=6). In 12 patients with a new clinically significant extracardiac finding, 6 (50%) experienced a change in care (medical therapy, n=4; palliative care, n=2). Conclusions CMR imaging identifies new clinically significant cardiac and noncardiac findings in half of patients with recent ESUS. Advanced cardiovascular screening should be considered in patients with ESUS. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT04555538.

Abstract Mechanistic cardiac electrophysiology models allow for personalized simulations of the electrical activity in the heart and the ensuing electrocardiogram (ECG) on the body surface. As such, synthetic signals possess known ground truth labels of the underlying disease and can be employed for validation of machine learning ECG analysis tools in addition to clinical signals. Recently, synthetic ECGs were used to enrich sparse clinical data or even replace them completely during training leading to improved performance on real-world clinical test data. We thus generated a novel synthetic database comprising a total of 16,900 12 lead ECGs based on electrophysiological simulations equally distributed into healthy control and 7 pathology classes. The pathological case of myocardial infraction had 6 sub-classes. A comparison of extracted features between the virtual cohort and a publicly available clinical ECG database demonstrated that the synthetic signals represent clinical ECGs for healthy and pathological subpopulations with high fidelity. The ECG database is split into training, validation, and test folds for development and objective assessment of novel machine learning algorithms.

Osteoarthritis (OA) is a chronic joint disease characterized by irreversible cartilage degradation. Current clinical treatment options lack effective pharmaceutical interventions targeting the disease’s root causes. MMP (matrix metalloproteinase) inhibitors represent a new approach to slowing OA progression by addressing cartilage degradation mechanisms. However, very few drugs within this class are in preclinical or clinical trial phases. Hydrogel-based 3D in vitro models have shown promise as preclinical testing platforms due to their resemblance to native extracellular matrix (ECM), abundant availability, and ease of use. Metalloproteinase-13 (MMP-13) is thought to be a major contributor to the degradation of articular cartilage in OA by aggressively breaking down type II collagen. This study focused on testing MMP-13 inhibitors using a GelMA-alginate hydrogel-based OA model induced by cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). The results demonstrate a significant inhibition of type II collagen breakdown by measuring C2C concentration using ELISA after treatment with MMP-13 inhibitors. However, inconsistencies in human cartilage explant samples led to inconclusive results. Nonetheless, the study highlights the GelMA-alginate hydrogel-based OA model as an alternative to human-sourced cartilage explants for in vitro drug screening.

Atrial fibrillation, the most prevalent cardiac arrhythmia, is treated by catheter ablation to isolate electrical triggers. Clinical trials on robotic catheter systems hold promise for improving the safety and efficacy of the procedure. However, expense and proprietary designs hinder accessibility to such systems. This paper details an open-source, modular, three-degree-of-freedom robotic platform for teleoperating commercial ablation catheters through joystick navigation. We also demonstrate a catheter-agnostic handle interface permitting customization with commercial catheters. Collaborating clinicians performed benchtop targeting trials, comparing manual and robotic catheter navigation performance. The robot reduced task duration by 1.59 s across participants and five trials. Validation through mean motion jerk analysis revealed 35.2% smoother robotic navigation for experts (≥10 years experience) compared to the intermediate group. Yet, both groups achieved smoother robot motion relative to the manual approach, with the experts and intermediates exhibiting 42.2% and 13.6% improvements, respectively. These results highlight the potential of this system for enhancing catheter-based procedures. The source code and designs of CardioXplorer have been made publicly available to lower boundaries and drive innovations that enhance procedure efficacy beyond human capabilities.

BackgroundOnchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease.Methodology/principal findingsCurrent World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay.Conclusions/significanceGiven the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

Summary: Background: Magnetic Resonance (MR) imaging is key for investigation of suspected newborn brain abnormalities. Access is limited in low-resource settings and challenging in infants needing intensive care. Portable ultralow field (ULF) MRI is showing promise in bedside adult brain imaging. Use in infants and children has been limited as brain-tissue composition differences necessitate sequence modification. The aim of this study was to develop neonatal-specific ULF structural sequences and test these across a range of gestational maturities and pathologies to inform future validation studies. Methods: Prospective cohort study within a UK neonatal specialist referral centre. Infants undergoing 3T MRI were recruited for paired ULF (64mT) portable MRI by convenience sampling from the neonatal unit and post-natal ward. Key inclusion criteria: 1) Infants with risk or suspicion of brain abnormality, or 2) preterm and term infants without suspicion of major genetic, chromosomal or neurological abnormality. Exclusions: presence of contra-indication for MR scanning. ULF sequence parameters were optimised for neonatal brain-tissues by iterative and explorative design. Neuroanatomic and pathologic features were compared by unblinded review, informing optimisation of subsequent sequence generations in a step-wise manner. Main outcome: visual identification of healthy and abnormal brain tissues/structures. ULF MR spectroscopy, diffusion, susceptibility weighted imaging, arteriography, and venography require pre-clinical technical development and have not been tested. Findings: Between September 23, 2021 and October 25, 2022, 102 paired scans were acquired in 87 infants; 1.17 paired scans per infant. Median age 9 days, median postmenstrual age 40+2 weeks (range: 31+3–53+4). Infants had a range of intensive care requirements. No adverse events observed. Optimised ULF sequences can visualise key neuroanatomy and brain abnormalities. In finalised neonatal sequences: T2w imaging distinguished grey and white matter (7/7 infants), ventricles (7/7), pituitary tissue (5/7), corpus callosum (7/7) and optic nerves (7/7). Signal congruence was seen within the posterior limb of the internal capsule in 10/11 infants on finalised T1w scans. In addition, brain abnormalities visualised on ULF optimised sequences have similar MR signal patterns to 3T imaging, including injury secondary to infarction (6/6 infants on T2w scans), hypoxia-ischaemia (abnormal signal in basal ganglia, thalami and white matter 2/2 infants on T2w scans, cortical highlighting 1/1 infant on T1w scan), and congenital malformations: polymicrogyria 3/3, absent corpus callosum 2/2, and vermian hypoplasia 3/3 infants on T2w scans. Sequences are susceptible to motion corruption, noise, and ULF artefact. Non-identified pathologies were small or subtle. Interpretation: On unblinded review, optimised portable MR can provide sufficient contrast, signal, and resolution for neuroanatomical identification and detection of a range of clinically important abnormalities. Blinded validation studies are now warranted. Funding: The Bill and Melinda Gates Foundation, the MRC, the Wellcome/EPSRC Centre for Medical Engineering, the MRC Centre for Neurodevelopmental Disorders, and the National Institute for Health Research (NIHR) Biomedical Research Centres based at Guy’s and St Thomas’ and South London & Maudsley NHS Foundation Trusts and King's College London.

Gelatin methacrylate (GelMA) is a photocrosslinkable biomaterial that has gained widespread use in tissue engineering due to its favorable biological attributes and customizable physical and mechanical traits. While GelMA is compatible with various cell types, distinct cellular responses are observed within GelMA hydrogels. As such, tailoring hydrogels for specific applications has become imperative. Thus, our objective was to develop GelMA hydrogels tailored to enhance cell viability specifically for TC28a2 chondrocytes in a three-dimensional (3D) cell culture setting. We investigated GelMA synthesis using PBS and 0.25M CB buffer, analyzed the mechanical and physical traits of GelMA hydrogels, and evaluated how varying GelMA crosslinking conditions (GelMA concentration, photoinitiator concentration, and UV exposure time) affected the viability of TC28a2 chondrocytes. The results revealed that GelMA synthesis using 0.25M CB buffer led to a greater degree of methacrylation compared to PBS buffer, and the LAP photoinitiator demonstrated superior efficacy for GelMA gelation compared to Irgacure 2959. Additionally, the stiffness, porosity, and swelling degree of GelMA hydrogels were predominantly affected by GelMA concentration, while cell viability was impacted by all crosslinking conditions, decreasing notably with increasing GelMA concentration, photoinitiator concentration, and UV exposure time. This study facilitated the optimization of crosslinking conditions to enhance cell viability within GelMA hydrogels, a critical aspect for diverse biomedical applications.

Atrial fibrillation (AF) underlies almost one third of all ischaemic strokes, with the left atrial appendage (LAA) identified as the primary thromboembolic source. Current stroke risk stratification approaches, such as the CHA2DS2-VASc score, rely mostly on clinical comorbidities, rather than thrombogenic mechanisms such as blood stasis, hypercoagulability and endothelial dysfunction—known as Virchow’s triad. While detection of AF-related thrombi is possible using established cardiac imaging techniques, such as transoesophageal echocardiography, there is a growing need to reliably assess AF-patient thrombogenicity prior to thrombus formation. Over the past decade, cardiac imaging and image-based biophysical modelling have emerged as powerful tools for reproducing the mechanisms of thrombogenesis. Clinical imaging modalities such as cardiac computed tomography, magnetic resonance and echocardiographic techniques can measure blood flow velocities and identify LA fibrosis (an indicator of endothelial dysfunction), but imaging remains limited in its ability to assess blood coagulation dynamics. In-silico cardiac modelling tools—such as computational fluid dynamics for blood flow, reaction-diffusion-convection equations to mimic the coagulation cascade, and surrogate flow metrics associated with endothelial damage—have grown in prevalence and advanced mechanistic understanding of thrombogenesis. However, neither technique alone can fully elucidate thrombogenicity in AF. In future, combining cardiac imaging with in-silico modelling and integrating machine learning approaches for rapid results directly from imaging data will require development under a rigorous framework of verification and clinical validation, but may pave the way towards enhanced personalised stroke risk stratification in the growing population of AF patients. This Review will focus on the significant progress in these fields.

BackgroundMansonella perstans is among the most neglected of the neglected tropical diseases and is believed to cause more human infections than any other filarial pathogen in Africa. Based largely upon assumptions of limited infection-associated morbidity, this pathogen remains understudied, and many basic questions pertaining to its pathogenicity, distribution, prevalence, and vector-host relationships remain unanswered. However, in recent years, mounting evidence of the potential for increased Mansonella infection-associated disease has sparked a renewal in research interest. This, in turn, has produced a need for improved diagnostics, capable of providing more accurate pictures of infection prevalence, pathogen distribution, and vector-host interactions.Methodology/principal findingsUtilizing a previously described pipeline for the discovery of optimal molecular diagnostic targets, we identified a repetitive DNA sequence, and developed a corresponding assay, which allows for the sensitive and species-specific identification of M. perstans in human blood samples. Testing also demonstrated the ability to utilize this assay for the detection of M. perstans in field-collected mosquito samples. When testing both sample types, our repeat-targeting index assay outperformed a ribosomal sequence-targeting reference assay, facilitating the identification of additional M. perstans-positive samples falsely characterized as "negative" using the less sensitive detection method.Conclusions/significanceThrough the development of an assay based upon the systematic identification of an optimal DNA target sequence, our novel diagnostic assay will provide programmatic efforts with a sensitive and specific testing platform that is capable of accurately mapping M. perstans infection and determining prevalence. Furthermore, with the added ability to identify the presence of M. perstans in mosquito samples, this assay will help to define our knowledge of the relationships that exist between this pathogen and the various geographically relevant mosquito species, which have been surmised to represent potential secondary vectors under certain conditions. Detection of M. perstans in mosquitoes will also demonstrate proof-of-concept for the mosquito-based monitoring of filarial pathogens not vectored primarily by mosquitoes, an approach expanding opportunities for integrated surveillance.

There is increasing evidence to suggest that atrial fibrillation is associated with a heightened risk of dementia. The mechanism of interaction is unclear. Atrial fibrillation-induced cerebral infarcts, hypoperfusion, systemic inflammation, and anticoagulant therapy-induced cerebral microbleeds, have been proposed to explain the link between these conditions. An understanding of the pathogenesis of atrial fibrillation-associated cognitive decline may enable the development of treatment strategies targeted towards the prevention of dementia in atrial fibrillation patients. The aim of this review is to explore the impact that existing atrial fibrillation treatment strategies may have on cognition and the putative mechanisms linking the two conditions. This review examines how components of the ‘Atrial Fibrillation Better Care pathway’ (stroke risk reduction, rhythm control, rate control, and risk factor management) may influence the trajectory of atrial fibrillation-associated cognitive decline. The requirements for further prospective studies to understand the mechanistic link between atrial fibrillation and dementia and to develop treatment strategies targeted towards the prevention of atrial fibrillation-associated cognitive decline, are highlighted.

Summary: The integration of neuroimaging and transcriptomics data, Imaging Transcriptomics, is becoming increasingly popular but standardized workflows for its implementation are still lacking. We describe the Imaging Transcriptomics toolbox, a new package that implements a full imaging transcriptomics pipeline using a user-friendly, command line interface. This toolbox allows the user to identify patterns of gene expression which correlates with a specific neuroimaging phenotype and perform gene set enrichment analyses to inform the biological interpretation of the findings using up-to-date methods.For complete details on the use and execution of this protocol, please refer to Martins et al. (2021).

Abstract Approximately one‐third of ischemic strokes are classified as cryptogenic strokes. The risk of stroke recurrence in these patients is significantly elevated with up to one‐third of patients with cryptogenic stroke experiencing a further stroke within 10 years. While anticoagulation is the mainstay of treatment for secondary stroke prevention in the context of documented atrial fibrillation (AF), it is estimated that up to 25% of patients with cryptogenic stroke have undiagnosed AF. Furthermore, the historical acceptance of a causal relationship between AF and stroke has recently come under scrutiny, with evidence to suggest that embolic stroke risk may be elevated even in the absence of documented atrial fibrillation attributable to the presence of electrical and structural changes constituting an atrial cardiomyopathy. More recently, the term embolic stroke of unknown source has garnered increasing interest as a subset of patients with cryptogenic stroke in whom a minimum set of diagnostic investigations has been performed, and a nonlacunar infarct highly suspicious of embolic etiology is suspected but in the absence of an identifiable secondary cause of stroke. The ongoing ARCADIA (Atrial Cardiopathy and Antithrombotic Drugs in Prevention After Cryptogenic Stroke) randomized trial and ATTICUS (Apixiban for Treatment of Embolic Stroke of Undetermined Source) study seek to further define this novel term. This review summarizes the relationship between AF, embolic stroke, and atrial cardiomyopathy and provides an overview of the clinical relevance of cardiac imaging, electrocardiographic, and serum biomarkers in the assessment of AF and secondary stroke risk. The implications of these findings on therapeutic considerations is considered and gaps in the literature identified as areas for future study in risk stratifying this cohort of patients.