The implementation of cancer molecular characterization in clinical practice has improved prognostic re-definition, extending the eligibility to a continuously increasing number of targeted treatments. Broad molecular profiling technologies better than organ-based approaches are believed to serve such dynamic purposes. We here present the workflow our institution adopted to run a comprehensive cancer genome profiling in clinical practice. This article describes the workflow designed to make a comprehensive cancer genome profiling program feasible and sustainable in a large-volume referral hospital. The introduction of molecular tumor profiling in the management of cancer patients is progressively forcing a reappraisal of the approach to cancer diagnosis and care.The possibility of prognostic re-definition and the availability of an increasing number of biomarker-driven targeted therapies have made the implementation of molecular characterization into clinical practice an essential need. Most of the advances in precision oncology rely on genomic sequencing and in particular on next-generation sequencing (NGS) approaches.The objective of personalized/precision medicine is the application of genomic information in order to define appropriate interventions  that could benefit both patients and health authorities in terms of clinical and healthcare outcomes .In 2020, the European Society of Medical Oncology (ESMO) recommended the implementation of comprehensive cancer genome profiling (CGP) for selected tumor types at least in academic centers .Moreover, discrepancies among different NGS assays, the lack of standardized operative procedures and the heterogeneity among clinical frameworks are major issues to be addressed in order to assess the real value of genomic testing for precision medicine.Aware of all these issues, the Fondazione Policlinico Universitario Agostino Gemelli IRCCS (FPG), a referral Italian research hospital, launched a CGP program  including 10 different cancer types. The program extends genomic assessment to more than 500 genes in a streamlined in-house process at no extra cost to the public healthcare system.Measurable outcomes include the feasibility of CGP from formalin-fixed paraffin-embedded (FFPE) and cytological specimens, turnaround time (TAT), time to therapy initiation, access to available target treatment (indication and off-label), enrollment in clinical trials and the rate of indications for referral to genetic counseling.The hybrid nature (diagnostic and research) of FPG500 might represent a blueprint for healthcare optimization in which a clinical process is turned into an opportunity for research to improve the care of cancer patients in the future. Patient, clinician and researcher journeys are shown in Patients can be included if affected by malignancies for which molecular characterization should be warranted according to national and international guidelines in specific clinical settings , colorectal, thyroid, endometrial cancers) and for which reimbursement policies are already in place in Italy see .Patients are selected during the multidisciplinary tumor board (MDT) sessions but are also referred by clinicians outside FPG. A molecular care manager has been selected to attend MDT meetings and together with referral oncologists for each cancer type is in charge of connecting patients, clinicians and study coordinators, smoothing the whole process. Patients are informed that the primary aim of CGP is to assess the presence of targetable mutations for which target therapies are already available as well as additional genomic alterations which might be relevant for research purposes. Moreover, variants associated with the risk of hereditary tumors can be found and will need subsequent confirmation of germline origin. All documentation  is provided by email and revised by the program coordinator before enrolment. Updates regarding patient enrolment are provided every week by email to clinicians, study coordinators and care managers to ensure continuous communication. Finally, weekly meetings are held among the whole team to discuss major and minor issues to be addressed.The whole analytical process (wet and dry steps) is managed through an all-in-one digital platform. The SLIMS (Agilent), designed for NGS, combines a laboratory information management system (LIMS) and an electronic laboratory notebook (ELN) to ensure data tracking and managing. In this workflow, all samples, from DNA/RNA extraction to sequencing, are tracked in an intuitive interface, and all laboratory protocols are integrated in order to follow each step of sample processing. The SLIMS is also integrated with instruments such as the Microlab STAR-Hamilton, for automated library preparation, and the Illumina Novaseq6000 for the sequencing. The platform has been further customized to be fully integrated with the hospital information system (SIO). In detail, input and output data between the SLIMS and the SIO are exchanged through Health Level Seven (HL7) messages, the most widely used messaging standard for the exchange of patient care and clinical information.Samples from surgeries, core needle biopsies, fine needle aspiration or cytology are reviewed by dedicated pathologists in order to assess tumor cell (TC) fraction. The minimum requirement to access nucleic acid extraction is a TC of at least 20%, with an optimal value being >30%.Once samples have been selected, Systematized Nomenclature of Medicine (SNOMED) codes for diagnosis and specimens are assigned on the dedicated platform. All H&E slices undergo digitalization before nucleic acid extraction.A semi-automated process takes place for simultaneous DNA/RNA extraction , DNA fragmentation , DNA/RNA quantification/qualification , automated library preparation  and sequencing (Novaseq6000-Illumina).Profiling is performed with the TruSight Oncology 500 high throughput , an assay that analyzes both DNA and RNA, identifying single nucleotide variants (SNVs), insertions/deletions (indels) and copy number variations (CNVs) in 523 genes as well as known and unknown fusions and splicing variants in 55 genes. In addition, it provides genomic “signatures” such as microsatellite instability (MSI) and tumor mutational burden (TMB), which is a measure of the total number of somatic mutations present in the sequenced genome. A validation process of the workflow was successfully run before implementing the test in the program.Samples not reaching the required quantity threshold for TSO500HT (DNA or RNA ≥ 40 ng) and for which no other specimens are available undergo Oncomine Focus Assay (OFA)  and Archer’s FusionPlex Lung Panel (AFL)  for DNA and RNA evaluation, respectively.Samples not reaching 20 ng of DNA/RNA are discussed with referral clinicians to evaluate either a re-biopsy or testing with standard-of-care techniques limited to the biomarkers approved for clinical practice and included in the Essential Levels of Assistance (LEA). Liquid biopsy is considered when no other option is available.https://github.com/lucianogiaco/lianne accessed on 09 August 2022), are sent to the Clinical Genomics Workspace software platform by Pierian Dx for variant interpretation and reporting.For TSO500HT, raw sequencing data are processed by the Illumina Software TSO500 v2.2 Local App and then, through a customized analysis pipeline . Afterward, the report is generated and data are filtered for non-synonymous, exonic variants and splice site variants in the flanking regions showing an allele frequency > 5%. Variants with a population minor allele frequency of more than 1% in 1000 Genomes and dbSNP are excluded since they are considered known polymorphisms.After sequencing, an accurate quality control (QC) at nucleotide resolution is performed using a custom tool integrated in the bioinformatic pipeline  nomenclature  and clasFor samples not fulfilling the requirements for TSO500 sequencing, targeted panels requiring a lower DNA/RNA input are used. The Oncomine Focus Assay (OFA) is a targeted NGS assay that enables the simultaneous detection of multiple variants across 35 tumor-related genes from DNA using amplicon-based enrichment. Library preparation, amplification and ligation steps are performed in line with the OFA protocol. Sequencing is performed using semiconductor sequencing technology . Data analysis is performed using the Torrent Server Variant Caller and the Ion Reporter Software .The Archer Fusion Lung (AFL) NGS assay is designed to detect key fusions in 17 genes, skipping events in EGFR vIII and MET exon 14, and select point mutations in 14 key lung cancer-associated genes. The AFL uses Archer’s Anchor Multiplex PCR chemistry to target regions of interest. Because of the use of one gene-specific primer and one universal primer, both known and unknown gene fusion partners can be detected. Reagent preparation and DNA synthesis, ligation and amplification are performed according to the official assay protocol.Libraries are multiplexed for sequencing on an Illumina MiSeq. Data are analyzed with the Archer Analysis software.The genomic report is reviewed by molecular biologists, bioinformaticians, pathologists and geneticists. When somatic variants have a possible germline correlation, genetic counseling is indicated. Specifically, patients will be referred to genetic assessment when a pathogenic or likely pathogenic variant with variant allele frequency (VAF) > 20% is identified in a clinically actionable cancer predisposition gene. Variants in genes known to be common targets of somatic hits, such as TP53 in all tumors or PTEN in endometrial carcinoma, will not be considered for genetic counseling unless suggested by clinical and/or molecular characteristics and family history ,66.An institutional Molecular Tumor Board (MTB) is available to help clinicians interpret and manage complex genomic profiling reports. In particular, the MTB discusses all cases with documented variants for which no approved drugs exist to verify the availability of clinical trials or off-label use of drugs . The MTB is scheduled every 2 weeks and involves not only a core team  but also optional professionals  included for case-specific discussions. The attending physician presents the case, and the board expresses a recommendation that is uploaded into the patient’s electronic chart.Clinical, family history, radiological, pathological, therapeutic and follow-up data for each enrolled patient are collected in a dedicated electronic case report form (eCRF) ,68. AcceThe majority of available CGP solutions are not In Vitro Diagnostics (IVD)-marked by Conformité Européenne (CE); this mark not only guarantees standards for quality and efficacy but is also required for reimbursement of diagnostic tests by many European health authorities. Academic centers can use non-IVD solutions if internally validated, benefitting also from the use of high-performance benchtop sequencing platforms which are not yet IVD-marked . As a maThe complexity and the high costs of adopting an in-house advanced genomic platform and dedicated team for data interpretation make the spread of genomics laboratories in healthcare institutions quite unlikely, even in privileged countries. In this scenario, a smooth, fast and safe transfer of patient samples and data should be planned across institutions and regions. A centralization of advanced genomics diagnostics in a sort of hub and spoke model would also allow efficient monitoring from health authorities encouraging accreditation processes in compliance with the EU General Data Protection Regulation (GDPR).High-quality clinical genomic data registries should be pursued by health authorities in order to evaluate the real impact of CGP in oncology. Cross-test comparison and validation tests to confirm mutations should be encouraged to evaluate the most cost-effective and efficient solutions available.Alternative strategies in case of low-quality or low-quantity material should be implemented. Referral centers should conduct studies dedicated to reinforcing evidence on the feasibility and reliability of liquid biopsy, which is more feasible in clinical practice, and to monitoring the evolution of the disease at several time points.Therapeutic and clinical implications of CGP for patients remain critical issues. Dedicated resources and favorable policies to fast-track research advances into clinical practice are required. Moreover, software dedicated to variant calling and clinical trial matching should be improved and customized based on local needs and national/international regulation authorities’ policies.Educational programs in genomics for healthcare professionals and physicians involved in cancer care should be integrated into training curricula.The availability of an MTB is fundamental for transversal education and for sharing therapeutic decisions in a multidisciplinary context.The identification of variants potentially associated with hereditary conditions is a plus for cancer prevention, but successful management of incidental findings including access to genetic counseling takes time and requires additional in-person consultations for dedicated blood sampling. It could be faster and more effective to confirm the germline origin of variants immediately after their identification by taking a blood sample at the time of enrollment. A study amendment on this topic has recently been considered and will soon be discussed.Economic resources should be dedicated to the automation of the large-scale sequencing process. This could not only reduce variability but also significantly reduce timing. CGP in fact allows almost the whole picture of genomic alterations to be obtained at once but takes much longer than targeted assays. Integrating CGP within patients’ diagnostic workup could be challenging for an optimal time to therapy initiation. Patient clinical conditions should always guide physicians to the most appropriate diagnostic option .Strategies for genomics data storage should be improved to avoid a bottleneck in the implementations of testing.Availability of and accessibility to targeted agents is a mainstay of the global process and the principal output from the clinical patient perspective.Major challenges to the implementation of genomic profiling into routine care have been outlined and only partially addressed in this program; they are detailed as follows:It seems clear that a sustainable and effective implementation of CPG within the diagnostic workup of cancer patients is challenging and requires improved policies and processes to concretely maximize benefits for present and future oncological patients and the healthcare system.


Using an implementation science framework, we detail how a national system for covering both standard and telemedicine laboratory testing would support a national PrEP program. Implementation strategies that will facilitate success include minimizing provider burden through uncomplicated billing systems and minimizing patient burden through centralized, online access systems. We anticipate that providing telemedicine and in-person laboratory testing options will optimize PrEP care by making it less burdensome, leading to cost-effective healthcare and improved population health. HIV pre-exposure prophylaxis provides a new opportunity to address the HIV epidemic in the United States. There were over 227,000 persons using PrEP in 2019,Developing structural support for individuals to practice healthy behaviors is a foundation of public health practice. Clinically-based prevention interventions, such as preventive medications or disease screenings, are often underutilized. This is understandable, especially when people do not have symptoms, when the benefits of prevention behaviors are dependent on an unknown future probability of disease acquisition, and when there are substantial financial costs of clinical prevention services.The need to optimize promotion of clinical prevention interventions drove the creation of the US Preventive Services Task Force in 1984, an independent panel that reviews and grades evidence for clinical prevention services.Coverage of laboratory and clinician costs removes a central barrier to PrEP care, because laboratory costs of PrEP are high. Using the laboratory fee reimbursement schedule from CMSThere are many ways to cover PrEP laboratory service gaps faced by many people in need of prevention services. We suggest developing a two-track system — in-person and telehealth care — that would allow the most users to initiate and remain on PrEP. Moreover, we do not anticipate that covering both options would increase the per user cost of the system; instead, telehealth services may potentially be cost-saving given that both lab and facility costs may be lower for telemedicine. Implementation science frameworks and change strategies should be incorporated into a national system to cover PrEP laboratory services.Despite the supportive USPSTF recommendation for PrEP, the no cost-sharing benefit does not apply to all persons in the United States. There remain substantial gaps that leave some of the persons hoping to use PrEP with substantial out-of-pocket costs, discouraging PrEP seeking and maintenance in care.For up to 48.9% of the US population, the ACA’s preventive services coverage and cost-sharing requirements are not federally required to be followed. Plans not federally required to follow CMS guidance include grandfathered healthcare plans (23.7 million persons),State Medicaid plans cover the cost of PrEP medication, but states may cover ancillary laboratory and clinician services at varying levels,An array of programs have been developed to fill gaps in PrEP ancillary services and drug coverage, including drug manufacturer assistance programs, state PrEP assistance programs, and usage of 340b drug program returns for service subsidy. There may be differential access to these services by place, program eligibility, and over time . This changing landscape of reimbursement makes navigating these different coverage rules incredibly challenging. In fact, most large PrEP prescribers have dedicated “navigators” to help patients seek coverage for their PrEP care across the various sources of funding.This patchwork system leads to considerable uncertainty for healthcare consumers and providers, likely deterring consumers from seeking PrEP and providers from prescribing it. In fact, PrEP usage is 99% greater in states that have expanded Medicaid and provide PrEP services coverage through state-level fee coverage programs that frequently feature PrEP navigation.The complexity of service coverage is even higher with the recent FDA approval of injectable cabotegravir.A national PrEP program, such as that proposed by Killelea and colleagues, could fill in PrEP laboratory coverage gaps, ensuring costs do not prevent those at risk from seeking PrEP care. Moreover, such a system could incorporate considerations of new PrEP regimens as they come to market. A recent cost-effectiveness analysis found that when PrEP drug costs are low, PrEP is a cost-saving intervention.A national PrEP services program, such as that proposed by Killelea and colleagues, will require laboratory support services that accommodate an array of patients and providers. We detail a patient-centered national service to cover PrEP laboratory services, proposing complementary models of in-person and telemedicine services to facilitate numerous PrEP access avenues. Using the Expert Recommendations for Implementing Change (ERIC) strategies,Standard care laboratory services could be covered with direct contracts with major laboratory services providers, or with a standard series of reimbursement payments for eligible laboratories. ERIC strategies to enhance provider prescription practices should include arrangements to ease billing by using existing channels and monitoring of service provision quality through ongoing surveys of patients and stakeholder interviews with providers to ensure services minimize provider burden. Audits should use PrEP utilization metrics and targets that feature not only assessments of overall use, but use according to equity. The PrEP-to-need ratioTelehealth laboratory services to promote PrEP are critical to provide primarily due to the high patient burden of four in-person clinic/lab visits per year, and to optimally serve those in need of PrEP who may be distant from PrEP providers such as those in rural areas.In our studies of home care for PrEP, participants have successfully self-collected specimens for each of these tests, meeting CLIA validation performance for laboratory developed tests and with the procedure receiving high acceptability ratings from persons in PrEP care. We anticipate that remote testing would not add costs beyond standard laboratory testing; in our experience, the costs of remote laboratory testing for PrEP, including mailing and materials, are lower than standard laboratory fees.ERIC strategies provide a number of insights into how to make a national telehealth laboratory program successful. To optimize telehealth benefits, online clinician services should be covered for persons otherwise facing a PrEP gap. A centralized program website could facilitate PrEP care initiation with referral to online providers that meet program and state medical licensing requirements. Systems to facilitate Electronic Health Records (EHR) transfer, from centralized mail-in laboratories to recipient clinics, would assist scale-up of telemedicine at traditional clinics that are interested in participating in the program.Audits should ensure that telehealth services are being provided to the same standard as in-person PrEP services, and should incorporate equity metrics to ensure program scale-up to communities most in-need of PrEP care to optimize the societal prevention impact of PrEP provision.There are many ways to cover PrEP laboratory service gaps faced by many people in need of prevention services. We suggest developing a two-track system — in-person and telehealth care — that would allow the most users to initiate and remain on PrEP. Moreover, we do not anticipate that covering both options would increase the per user cost of the system; instead, telehealth services may potentially be cost-saving given that both lab and facility costs may be lower for telemedicine. Implementation science frameworks and change strategies should be incorporated into a national system to cover PrEP laboratory services.Future discussions and development of a national PrEP coverage system should consider these concepts and be informed by appropriate frameworks for implementation science to ensure a thorough exploration that seeks to minimize patient-level barriers to accessing PrEP.


Gadolinium-enhanced cardiac magnetic resonance has revolutionized cardiac imaging in the last two decades and has emerged as an essential and powerful tool for the characterization and treatment guidance of a wide range of cardiovascular diseases. However, due to the high prevalence of chronic renal dysfunction in patients with cardiovascular conditions, the risk of nephrogenic systemic fibrosis (NSF) after gadolinium exposure has been a permanent concern. Even though the newer macrocyclic agents have proven to be much safer in patients with chronic kidney disease and end-stage renal failure, clinicians must fully understand the clinical characteristics and risk factors of this devastating pathology and maintain a high degree of suspicion to prevent and recognize it. This review aimed to summarize the existing evidence regarding the physiopathology, clinical manifestations, diagnosis, and prevention of NSF related to the use of gadolinium-based contrast agents. Magnetic resonance imaging (MRI) contrast agents serve to improve diagnostic images’ sensitivity and specificity by altering the tissues’ intrinsic properties. Contrast agents carry strong paramagnetic properties that can be exploited to provide enhanced contrast between healthy and diseased tissues. By shortening the T1 and T2 relaxation times of the contiguous hydrogen nuclei, paramagnetic elements such as gadolinium (Gd) enhance the soft tissue contrast and help characterize a wide array of pathologies ..1].Most GBCAs have preferential renal elimination, and their clearance is highly dependent on the glomerular filtration rate. A small percentage of the administered dose is eliminated via the hepatobiliary route. In CKD patients, the prolonged GBCAs’ half-life leads to a significant release of Gd, and therefore, a higher burden of Gd precipitates. Moreover, some of the anions that are thought to play a critical role in transmetalation (such as phosphates) are often elevated in CKD patients, facilitating this pathologic process by increasing the substrate availability for compound formation ,27.2) or acute renal insufficiency of any severity due to hepato-renal syndrome or in the perioperative period after liver transplantation. In 2010, the European Medicines Agency (EMA) released a statement in which NSF was considered a potential side effect of GBCAs based on the number of published reports . On. On2) orAcute kidney injury: the presence of AKI significantly increases the risk of NSF ,39,47. IIn those patients with terminal CKD already on RRT , dialysis should continue after receiving a GBCA. GBCA infusion should be performed as closely before hemodialysis as is possible . These pThere is insufficient evidence to support changing patients from peritoneal dialysis to hemodialysis or altering dialysis prescription after the infusion of a GBCA. Peritoneal dialysis may be less effective than hemodialysis in clearing circulating GBCA; however, there is no evidence regarding the superiority of a specific type of RTT in order to decrease the risk of NSF ,49.NSF occurs most commonly in patients who received high doses of GBCA, either as a single dose or cumulatively after multiple administrations . In some2), there is no contraindication if the examinations are determined to be necessary [In patients with preserved or moderately reduced kidney function  described in patients with severe renal impairment exposed to a gadolinium-based contrast agent.There is not a specific treatment or prophylaxis regimen to treat or prevent the onset of NSF.Even though the newer macrocyclic agents have proven to be much safer in patients with chronic kidney disease and end-stage renal failure, clinicians must fully understand the clinical characteristics and risk factors of this devastating pathology and maintain a high degree of suspicion to prevent and recognize it. Cardiac MRI with late gadolinium enhancement (LGE) has significantly impacted the management, decision making, and diagnosis of various cardiomyopathy or interstitial heart disease. However, the over-concerned about nephrogenic systemic fibrosis may make cardiac MRI with LGE be avoided inappropriately. The risk and benefits of this imaging study should be balanced.


South Africa’s malaria elimination plans are aligned to the World Health Organization’s aim for a malaria-free world and include specific objectives within a specified time frame. These are proving difficult to achieve owing to the sporadic nature of locally acquired malaria in some affected districts, while other districts that were endemic for the disease are either malaria-free or very close to that goal. The WHO also specifies that continued measures to prevent the re-establishment of transmission are required in areas where elimination has been achieved. These measures include routine malaria vector surveillance in endemic districts that are free of malaria to assess receptivity and risk of reintroduction, which may prove difficult to justify in the face of competing public health priorities and limited resources. These issues are discussed here within the framework of vector surveillance and control and include recommendations on how they can be addressed going forward. The World Health Organization defines malaria elimination as “the interruption of local transmission of a specified malaria parasite species in a defined geographical area as a result of deliberate activities” . FollowiMost malaria cases recorded in South Africa are imported, with low-level locally acquired malaria restricted to a comparatively small number of districts in the three endemic provinces: KwaZulu-Natal, Mpumalanga and Limpopo . These dPlasmodium falciparum (>97%) with very small contributions from P. ovale, P. malariae and P. vivax [Malaria in South Africa is primarily caused by P. vivax . Point-oiparum >9% with vePlasmodium parasite populations and insecticide resistance in Anopheles mosquito vector populations, which led to programmatic failure despite the implementation of interventions [The malaria incidence in South Africa is seasonal and tends to peak in late summer after significant rainfall ,6. Localventions ,9.Provision of effective management, leadership and coordination;Provision of sustainable and strengthened surveillance systems ensuring that 100% of cases are reported to the Malaria Information System (MIS);Community outreach and the provision of pertinent malaria information;Measures to ensure that all populations at risk receive at least 95% coverage with key vector suppression strategies and interventions;Universal access to diagnosis and treatment in endemic and non-endemic areas according to national guidelines.South Africa’s malaria elimination agenda for the period from 2019 to 2023 is based on five broad strategic objectives  built onAnopheles breeding sites complement the IRS campaigns [Vector control (objective 4) in the endemic provinces includes either blanket or targeted indoor residual spraying (IRS) of specially formulated insecticides  dependinampaigns . Cross-bampaigns ,4. Vectoampaigns ,10,11. TDespite annual vector control interventions, locally acquired malaria persists in South Africa. It is often characterized by an unusual epidemiological profile in low-incidence settings whereby cases are sporadic as opposed to ongoing or recurrent transmission characteristics of endemicity in higher incidence settings , such asA significant proportion of locally acquired malaria cases in South Africa are now classified as secondarily imported cases. These occur when local vector mosquitoes acquire malaria infections from infected (often asymptomatic) people who are migrants from or have recently travelled to a malaria-endemic area such as a neighboring country. This can lead to sporadic clusters of local malaria when infected mosquitoes subsequently bite local residents. An example is sporadic clusters of local cases on sugarcane farms in Zululand District during the planting and harvesting seasons.The importation of infective mosquitoes from neighboring countries or other endemic areas within South Africa is also a possible explanation for at least some of the sporadic locally acquired cases. Odyssean malaria  occurs via the inadvertent transfer of one or several infective mosquitoes via land or air transport. Such mosquitoes can alight from vehicles at some point during their journey from where they bite and infect one or more local residents who happen to be in close proximity to where the infective mosquito/es escaped . Although it is not possible to distinguish between odyssean and local malaria in endemic areas, a proxy for the occurrence of this category of malaria comes from its occurrence in non-endemic provinces  within South Africa, especially Gauteng Province. Between two and ten odyssean malaria cases are recorded in Gauteng Province annually, their classification being based on affected patients having no travel history to a malaria-affected area in the month preceding the onset of symptoms . If odysThe sporadic nature of local malaria incidences in many of the affected low-incidence districts is problematic for malaria elimination because it is difficult to plan targeted vector control interventions without having a reliable predictor of where future cases are most likely to occur. This can be addressed by the foci-clearing protocols that are based on a set of reactive measures when one or more cases occur. These measures include active case detection and treatment of any infected people resident in or in the vicinity of the index house, vector control in index houses and their immediate neighbors, larval source management in the vicinity of the index house and vector surveillance. It is, therefore, the author’s opinion that vector control measures, suitably implemented, can enable malaria elimination by reducing incidences to very low levels, but the actual elimination by districts then depends on parasite clearance by active and passive case detection.Plasmodium parasites via the movements of infected people. What matters is how receptive these areas are to malaria transmission [Some districts and municipalities in South Africa’s malaria-endemic provinces  are almost malaria-free in terms of local transmission. This does not, however, mean that they are safe from the reintroduction of malaria by the importation of smission .Anopheles mosquito. The only possible and extremely rare exception to this rule is the acquisition of malaria by blood transfusion. Assessing the risk of malaria in any given area or region is therefore based on the occurrence of Anopheles vector populations. These are generally classified as either major or secondary vectors via a loosely defined classification system based on their measured or inferred contributions to malaria transmission. Five Anopheles species are implicated as malaria vectors within South Africa’s borders [Anopheles funestus sensu stricto and An. arabiensis. These two species tend to associate closely with human communities  from whom they acquire most of their blood meals, and are, therefore, responsible for the bulk of locally acquired malaria cases. Secondary vectors include An. vaneedeni, An. parensis and An. merus. They are listed as secondary vector species either because they interact with human communities to a lesser extent than the major vectors, because they are rarer, or because they are less susceptible to Plasmodium infections and are, therefore, comparatively inefficient vectors. Secondary vectors can, however, play prominent roles in malaria transmission and should not be dismissed during vector control operational research and planning [Malaria can only be acquired through the bite of an infective  borders ,20,21. Tplanning .Anopheles species assemblage for the area in question, insecticide susceptibility profiles of each vector population, and information concerning their feeding (indoor or outdoor and peak biting times), resting (indoor or outdoor) and breeding characteristics (characteristics of water bodies that they choose for egg deposition). This information enables an assessment of the risk of malaria reintroduction, and can be used for planning vector control operations—i.e., appropriate methods and insecticide choice—should reintroduction occur.Assessing risk and receptivity in a malaria-free area that was previously endemic for the disease involves regular surveillance for Anopheles mosquitoes. This is a complex task requiring skilled entomologists who are able to employ a range of methods designed to catch adult and aquatic-stage Anopheles mosquitoes . ProcessImportantly, here there is the recognition of the need for two critical interventions: ongoing surveillance and control including outbreak response. Intensive malaria parasite and vector surveillance pre- and post-elimination will need to be maintained ,24. ManyLow-incidence districts earmarked for malaria elimination should ensure the full implementation of proactive or reactive vector control methods year on year, even after malaria elimination is achieved.Low incidence districts earmarked for malaria elimination should plan, implement and maintain active and passive case detection, especially amongst migrant and immigrant communities.Assessments of malaria risk and receptivity in malaria-free districts in endemic provinces should be routinely conducted by provincial entomological surveillance officers on an annual basis.Anopheles species assemblages and insecticide susceptibility profiles of known vector populations.Annual vector control plans at the local  level should be based on Additional vector control methods including dual active ingredient insecticide-treated bed net distribution, community outreach in terms of personal protection methods, housing design and screening, and environmental management (such as drainage of non-utilized water bodies used by mosquitoes for breeding) should be considered pre- and post-malaria elimination at the local level.Political commitment and the resources required for ongoing surveillance and control operations will need to be sourced year on year from national and provincial governments. This will require a multi-stakeholder approach coordinated by the NDoH Malaria Directorate. Important government department stakeholders include Agriculture, Finance and Environment.Cross-border initiatives need to be maintained, sustained and strengthened as needed, especially in terms of vector and parasite surveillance at border crossings/posts.


Introducing a Special Issue on mechanism of reproductive disorders in mammals originating from exposure to environmental chemicals during perinatal life. Exposure to chemicals during perinatal life can disrupt reproductive development and ultimately lead to reproductive disorders. This includes disorders manifesting already at birth such as genital malformations, but also late onset disorders such as reproductive cancers and infertility . The undBroadly speaking, male sexual differentiation depends on a surge in androgen signaling during critical stages of development, whereas female sexual differentiation requires androgen levels to be low during the same developmental stages. This is overly simplified, of course, as estrogens and other hormones also play essential roles in reproductive development of both sexes, as do other signaling pathways and biomolecules. Sexual development relies instead on a complex network of signaling pathways that interact to ensure proper spatiotemporal tissue differentiation and many of these signaling pathways may themselves be vulnerable to disruption by exogenous chemical substances.To better account for the many ways by which chemicals can disrupt reproductive development, this special issue invited contributions that could shed new light on causal molecular pathways in addition to classical sex hormone signaling. A more long-term vision is that such new insights can be used in a chemical safety assessment and ultimately regulatory decision-making. Especially in a new era of predictive toxicology where chemical risk assessors are asked to rely much more on non-animal test data than on classical animal toxicity studies when determining if a chemical substance may or may not pose a risk to human health.Disrupted steroid hormone signaling could account for a majority of effect modalities causing reproductive disorders through early-life exposure to chemicals. However, both gonadal differentiation and sexual development rely on additional signaling pathways, for instance the morphoregulatory pathways Wingless-like (WNT), retinoic acid (RA) and Hedgehog (HH). In the first article of this Special Issue, the HH signaling pathway is discussed in the context of reproductive development and disruption by environmental chemicals or drugs . AlthougThe third article uses the Adverse Outcome Pathway (AOP) framework to construct putative causal toxicological pathways linking developmental exposures to EDCs, particularly phthalates, with malformations of the male reproductive tract . The AOPAs a first case for this pragmatic approach to AOP development, the fourth article presents a key event relationship (KER) linking reduced RA signaling in the developing ovary with the failure of oocytes to enter meiosis . The RA Since modern toxicology is moving towards relying more on non-animal test strategies for safety assessments of chemical substances, it is imperative that we understand the underlying mechanisms and modes of action that underpin reproductive toxicity . Only thGiven his role as Guest Editor, Terje Svingen had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Thomas Knudsen.Terje Svingen: Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


N = 69 participants with aphasia following left hemisphere stroke) consisted of a diverse and comprehensive set of narrative speech production measures and measures of overall severity, semantic deficits, and phonological deficits. These measures were entered into a principal component analysis with bifactor rotation—a latent structure model where each item loads on a general factor that reflects what is common among the items, and orthogonal factors that explain variance not accounted for by the general factor. Lesion data were available for 58 of the participants, and each factor score was analysed with multivariate lesion–symptom mapping. Effects of connectivity disruption were evaluated using robust regression with tract disconnection or graph theoretic measures of connectivity as predictors. The principal component analysis produced a four-factor solution that accounted for 70.6% of the variance in the data, with a general factor corresponding to the overall severity and length and complexity of speech output (complexity factor), a lexical syntax factor, and independent factors for Semantics and Phonology. Deficits in the complexity of speech output were associated with a large temporo-parietal region, similar to overall aphasia severity. The lexical syntax factor was associated with damage in a relatively small set of fronto-parietal regions which may reflect the recruitment of control systems to support retrieval and correct usage of lexical items that primarily serve a syntactic rather than semantic function. Tract-based measures of connectivity disruption were not statistically associated with the deficit scores after controlling for overall lesion volume. Language network efficiency and average clustering coefficient within the language network were significantly associated with deficit scores after controlling for overall lesion volume. These results highlight overall severity as the critical contributor to fluent speech in post-stroke aphasia, with a dissociable factor corresponding to lexical syntax.Deficits in fluent speech production following left hemisphere stroke are a central concern because of their impact on patients’ lives and the insight they provide about the neural organization of language processing. Fluent speech production requires the rapid coordination of phonological, semantic, and syntactic processing, so this study examined how deficits in connected speech relate to these language sub-systems. Behavioural data  general impairment severity and sentence/utterance complexity, which are associated with damage in a large temporo-parietal region and (ii) lexical syntax, which is associated with damage in a relatively small set of fronto-parietal regions.Zevgolatakou   In the intervening 100 + years, it has become clear that fluent speech production requires the rapid coordination of multiple cognitive and neural systems, including syntax, sentence planning and working memory, semantics, and phonology and articulatory motor control.5-8 This study used data-driven methods to investigate the relationships among fluency, overall aphasia severity, and deficits of syntax, semantics and phonology.Aphasia is an acquired impairment of language production and comprehension that affects approximately one-third of stroke survivors and is one of the most frequent and debilitating consequences of brain injury.9-13 A key problem is that multiple different underlying impairments can disrupt fluent speech production. These underlying impairments reflect different cognitive sub-systems with different neural bases and may require different treatment approaches.10Although aphasia is traditionally divided into ‘fluent’ and ‘non-fluent’ subtypes, this distinction has been widely criticised for not correctly capturing symptom co-occurrence and for having a poor agreement between diagnostic instruments.14-16Deficits of planning or executing the articulatory gestures that make up speech can produce hesitations, phonological errors and distortions, which are one form of non-fluent speech. Such deficits can arise from impairments in phonological planning or in articulatory motor control (e.g. dysarthria) or their interface (apraxia of speech) and are associated with damage to precentral motor control regions, postcentral somatosensory cortex, and inferior parietal regions that support phonological–articulatory planning.18 Impairments of these lexical–semantic processes are associated with damage to anterior temporal and inferior frontal regions.19-21Difficulty with retrieving the intended word can also produce hesitations during a connected speech while the speaker searches for that word. Such difficulty results from impaired lexical–semantic processes: semantic knowledge of words and selection processes to choose among candidate words.23 Agrammatism is characterized by reduced or omitted syntactic structure, which is a common characteristic of non-fluent speech. These structural elements can be at the sentence-level  or at the lexical-level . A recent comprehensive framework integrated a large body of evidence to propose that posterior superior and middle temporal regions support hierarchical lexical–syntactic functions for production and comprehension while inferior frontal regions support morpho-syntactic sequencing primarily relevant for production.6 This view was supported by a dissociation between reduction/omission of grammatical structure (‘agrammatism’), which was associated with frontal damage, and grammatical errors that were not reductions (‘paragrammatism’), which was associated with posterior superior and middle temporal damage.24Syntactic processes are also critical for fluent speech production, and there is a long history of association between ‘agrammatism’ and non-fluent aphasia.25 In contrast to a syntax-specific view, some have argued that syntactic deficits are a result of relatively general cognitive resource reductions.26 At least one study7 found no regions where damage was associated with a syntactic deficit; it is likely that other such null results exist but have been suppressed by publication bias.However, the neural basis for syntactic processing is somewhat inconsistent across studies, implicating large regions of frontal, parietal, and temporal cortex, as well as the underlying white matter. Some evidence suggests that the language network shows lexical–semantic and combinatorial sensitivity with no regions selective for purely syntactic processes.27-30 More recent ‘network neuroscience’ work has quantified connectivity disruption using graph theory measures, which are meant to capture broader connectivity properties like the efficiency of information transmission and integration or segregation of neural components.31 One study found that graph theory measures outperformed connection weights as predictors of fluency deficits.32Thus, fluency deficits could plausibly arise from phonological/articulatory, lexical–semantic, or syntactic deficits. Connectivity within these systems also plays an important role: several studies have found that damage to frontal and peri-Sylvian white matter tracts is an important contributor to fluency deficits.33 A more focused examination of 226 participants with post-stroke aphasia found that a ‘mild aphasia’ cluster was behaviourally and neuroanatomically distinct from the other two clusters, which corresponded to the semantic deficit and phonological deficit.34A further complication is that recent studies have identified aphasia severity as a major dividing feature. A large-scale study of 330 participants with aphasia across three different aetiologies  found that severity was the primary dimension of variability, explaining 75% of the variance in aphasia battery sub-scores.35-37 . A key factor analysis study5 of spontaneous speech in 274 individuals with aphasia identified 6 latent factors that accounted for 52% of the variance, with substantial overlap between some of the factors. In particular, four of the factors were strongly correlated and primarily reflected fluency (words per minute) and syntactic structure . The other factors reflected semantic anomalies (e.g. jargon) and utterance repairs.Data-driven statistical methods like factor analysis and principal component analysis (PCA) use correlations among measures to group them into latent factors or components and calculate composite scores for those factors. Several studies have focused on measures of connected speech and used factor analysis to identify clusters of measures that suggest common underlying components40 found that frontal regions were associated with speech sound distortions and syntactic deficits, anterior and inferior temporal regions with lexical retrieval, and posterior temporal regions with phonological errors. In post-stroke aphasia, a statistical path modelling analysis8 found that syntactic impairment had a strong and direct association with fluency, while word production, comprehension and working memory deficits were indirectly associated with fluency.Because multiple cognitive sub-systems contribute to fluent speech production, it is important to consider them simultaneously. That is, combining measures derived from connected speech elicitation tasks with other measures that capture specific sub-systems is necessary for investigating how fluency relates to those sub-systems. A study of 50 participants with primary progressive aphasiaSuch multi-dimensional approaches can allow investigation of how the sub-systems relate to one another in addition to how they contribute to fluency. This is particularly important for the domain of syntax deficits, which (as briefly reviewed in the previous section) have sentence-level and word-level aspects, and could result from general resource reduction  or combinatorial deficits rather than syntax-specific impairments.39 In these studies, the fluency components had high loadings from composite measures of spontaneous speech production [e.g. fluency scores from aphasia assessments such as the Western Aphasia Battery (WAB) and Comprehensive Aphasia Test] and coarse measures of fluency (such as words per minute and mean length of utterance). A recent study41 used finer-grained discourse production measures and showed that connected speech production had distinct components of quantity , quality , and words per minute (which they labelled ‘motor speech ability’). Of note, these studies included varied measures of fluency but very limited measures of syntax. Another recent study specifically focused on connected speech production in acute left hemisphere stroke42 and found distinct components of utterance length/complexity, syntactic accuracy, lexical retrieval, and words per minute (which they labelled ‘production fluency’). This last study suggests dissociations between phonological, semantic, syntactic, and combinatorial processes, but the semantic measures were limited, and phonological measures were virtually absent, so the connection to those broader components remains unclear.Several recent studies have applied this approach and identified consistent dissociations between fluency, semantic, phonological, and executive deficits.23 to derive a diverse set of narrative speech production measures that include fluency and syntax at lexical, utterance and sentence levels. Like Ding et al.,42 we used a comprehensive set of QPA measures; however, we also included general measures of language impairment and measures of semantic and phonological deficits. These additional measures served as ‘anchors’ for three well-established aspects of aphasic deficits  allowing the QPA-based measures of fluency and syntax deficit to attach to those factors or to form separate factors.The current study used a data-driven approach to evaluate how measures of fluency and syntax deficits in post-stroke aphasia relate to overall aphasia severity, semantic deficits, and phonological deficits, and the lesion correlates of these components. We build on prior data-driven work, making several key innovations and improvements. First, we used quantitative production analysis (QPA)44 Because we included anchor variables and used bifactor rotation, we expected to find severity, semantic, and phonological factors; the key research question was how the QPA-based measures of fluency and syntax would distribute among those factors or form additional factors.Second, to explicitly capture overall severity as a core dimension of variability in aphasia, we used a bifactor PCA model. Bifactor modelling re-emerged relatively recently as a psychometric technique that hypothesizes a single general factor, which explains shared variance across all items (measures), and a series of orthogonal (uncorrelated) domain-specific factors.45 SCCAN is particularly important for the present study because fluency and syntax are likely to rely on a distributed network of brain regions, and SCCAN is better able to detect networks of symptom-relevant brain regions.45 In addition, SCCAN uses cross-validation (CV) to optimize the sparseness of the solution and evaluates the prediction accuracy  of the overall solution. As a result, the accuracy of lesion-deficit prediction determines the size of the critical region identified by the LSM analysis. In contrast, in mass-univariate LSM, the focality of the result is based on the conservativeness of the correction for multiple comparisons, which is set based on statistical considerations.46-48 Therefore, SCCAN is better able to capture distributed networks of lesion-relevant regions and to determine whether there are (possibly multiple) small symptom-relevant regions or a single large region.Third, we conducted lesion–symptom mapping (LSM) analyses using a multivariate optimization technique known as sparse canonical correlation analysis for neuroimaging (SCCAN), which more accurately identifies symptom-relevant lesion areas.50 in fluency and syntax deficits, although we did not find specific effects of frontal white matter damage in our prior work.7 That is, the current state of the evidence is equivocal, possibly because there is substantial variability in both the methods and results of prior work on effects of connectivity disruption on language deficits in post-stroke aphasia. Therefore, these exploratory analyses were intended to help build toward a consensus rather than to test specific hypotheses. In the current study, we evaluated the effects of damage to a larger set of language-relevant white matter tracts . We also evaluated the effects of connectivity disruption using measures derived from graph theory , which are meant to capture broader communication efficacy and efficiency.Fourth, based on the premise that connectivity disruption can undermine functional networks that support language production, we tested the contribution of white matter damage using both tract-based and network-based measures. This was motivated by prior studies that highlighted the role of white matter damage [particularly the frontal aslant tract (FAT) and the anterior portion of the arcuate fasciculus (AF)]In sum, the present study provides a comprehensive behavioural and neural assessment of fluency deficits in post-stroke aphasia. A data-driven approach was used to determine how a diverse set of measures of fluency and syntax processing were associated with or dissociated from measures of aphasia severity, semantic deficits, and phonological deficits. Multivariate LSM and connectivity disruption analyses were then used to determine the neural correlates of these behavioural deficit dimensions.51 We conducted a preliminary leave-one-out PCA stability analysis in which we iteratively removed an individual participant and tested the effect of that exclusion on the PCA results (this is a non-parametric version of standard outlier detection methods). In general, there was a very high correlation (>0.95) between PCA results with and without a participant, indicating stable results that are minimally influenced by any one participant. However, there were five high-influence participants: excluding one of these participants substantially changed the results. These five outlier participants were excluded from the analysis . The analytic sample contained behavioural data from 69 participants and imaging data were available for 58 of these participants.The initial data set consisted of behavioural data from 74 participants who were native English speakers, right-handed, and had suffered a left hemisphere stroke. These participants are the subset who completed the connected speech elicitation task from a larger study of post-stroke aphasia.53 A summary of demographic and clinical information is shown in All but five participants were examined at the chronic stage of aphasia (at least 3 months post-onset), but the exclusion of these five participants did not change the results so the more inclusive analyses are reported here. The data are part of a larger study on language processing following left hemisphere stroke and several previous articles on language deficits in aphasia have included subsets of the participants.N = 45) or CT (N = 13) brain scans, following the same procedures as previous studies of this data set.53 For the MRI scans, lesions were manually segmented on each participant’s T1-weighted structural image by a technician and reviewed by an experienced neurologist for accuracy. Each participant’s brain image was registered to the Montreal Neurological Institute space Colin27 template by an automated symmetric diffeomorphic registration algorithm,54 which iteratively samples the space to find the best solution to align the intact portion of the brain (i.e. with the lesion masked out) with the template. This solution (image transformation) is then applied to the lesion mask to register it to the same template. For the CT scans, a neurologist drew the lesions directly onto the Colin27 template after rotating it (pitch only) to match the approximate slice plane of the participant’s scan.Lesion location was assessed based on MRI  in order to reach at least 150 words. The speech samples were transcribed and coded by a speech pathologist or research assistant specifically trained to perform transcription and QPA coding following guidelines based on published QPA scoring protocol.56 the measures correspond to lexical content , auxiliary verb usage (auxiliary complexity index), and structural analysis . See 57 and a goal of this study was to evaluate how these measures relate to aphasia severity, semantic deficits and phonological deficits.The measures selected for the analyses reflected a wide range of morphological, structural and lexical properties of speech. Based on the grouping described in the QPA manual,The behavioural testing of the participants consisted of a wide range of neuropsychological batteries and tests. To supplement the QPA measures, a subset of measures was selected to reflect overall severity and two important contributors to language production that are not captured by QPA measures: phonology and semantics. These measures provide are optimized for capturing phonological and semantic deficits and are independent of connected speech, so they provide a stronger assessment of whether aspects of connected speech are a result of such deficits.WAB-Revised:58 A standard aphasia assessment battery that provides an overall measure of aphasia severity—the aphasia quotient (AQ). The measure used is WAB AQ.Philadelphia Naming Test (PNT):59 A test of single word production in object (picture) naming that provides measures of overall word production deficit (accuracy) as well as semantic and phonological deficits . Measures used are as follows: proportion of correct responses, proportion of semantic errors and proportion of formal and non-word errors.Camel and Cactus Test (CCT):60 A picture-based semantic association test. The measure used is percentage of correct responses.Semantic discrimination: A verbal semantic judgment task derived from the semantic category probe task61 by using only list-length 1 data. The measure used is percentage of correct responses.Philadelphia Repetition Test (PRT):62 A test of word repetition using the same words as the PNT. This test primarily measures phonological processing with lexical contributions.64 The measure used is percentage of correct responses.Non-word repetition task:61 A test of non-word repetition, which primarily measures phonological production. The measure used is percentage of correct responses.r > 0.98 for each of the four factors/components] and it is more straightforward to compute individual participant scores from PCA, so PCA was used here). A bifactor model is a latent structure model where each item loads on a general factor that reflects what is common among the items, and two or more orthogonal factors that potentially explain variance not accounted for by the general factor.44 In this case, overall aphasia severity plausibly affects all measures, so using a bifactor model explicitly allows all measures to load on this general factor. The measures that load strongly and only on this factor will be ones that are very closely related to aphasia severity. The additional factors can then represent language sub-systems that may be affected by stroke independently of overall severity. In other words, the bifactor model allows directly exploring the extent to which items reflect a common trait (aphasia severity) and the extent to which they reflect statistically independent sub-systems . The resulting component scores for every participant were then used for the LSM analysis.The 21 behavioural measures  were entered into a principal component analysis (PCA) with a bifactor rotation.  while minimizing the number of voxels in the solution (i.e. sparser solutions are favoured over less sparse ones). As a result, if a behavioural score can be predicted from lesion in a small subset of voxels, SCCAN will identify this subset of voxels; if damage throughout a large territory is associated with the behavioural score, then SCCAN will find that non-sparse solution.LSM analyses were performed using SCCAN.66), where statistical significance is evaluated at the level of individual voxels and requires correcting for multiple comparisons across many voxels. The conservativeness of this multiple comparisons correction has a major impact on the focality of results. Unlike these other LSM methods, the sparseness or focality of SCCAN LSM results is determined by predictive accuracy of the overall solution.The goodness of the SCCAN solution is the correlation between the predicted and observed behavioural scores and the statistical significance of that correlation is used to determine whether the SCCAN solution is acceptable. If SCCAN is not able to identify a set of voxels that statistically significantly predict the behavioural score, then SCCAN has failed to identify an adequate solution. Critically, the SCCAN solution is evaluated in terms of its overall predictive accuracy, not individual voxels within it, so there is only one overall statistical test, not individual tests for voxels. This is in stark contrast to mass-univariate LSM and some multivariate LSM methods . The toolkit calculates connectivity disruption by overlaying the lesion files on an atlas of intact connectivity.69 This whole-brain connectivity atlas was derived using deterministic tractography on the population-averaged HCP-842 data set with multiple turning angle thresholds to obtain 500 000 population-level streamline trajectories , which were manually vetted and assigned to known white matter fibre tracts by a team of neuroanatomists.69Connectivity disruption measures were derived using atlas-based metrics of lesion impact generated in MATLAB 2020bParticipant binarized and spatially normalized lesion files were used as inputs to the toolkit, which was run with the following parameters: connection criteria = pass, spared connection threshold = 50 and smoothing kernel = 2. The primary atlas-based measures of interest were (i) disconnection severity within critical left hemisphere white matter tracts, (ii) metrics describing the whole-brain network structure, and (iii) metrics describing the regional structure of a language sub-network.69 and quantifying the number of streamlines intersecting the lesion relative to the total number of streamlines within the tract. This method of estimating tract disconnection severity has been shown to be more sensitive than traditional lesion load measures.70 Disconnection severity within the left superior longitudinal fasciculus (SLF), left AF, left UF, left FAT, and left IFOF were used for analyses.Tract-based disconnection severity was calculated by overlaying the lesion files on each tract within the HCP-842 tractography atlas69 removing streamlines that intersected with the participant’s lesion, and counting the number of remaining streamlines that passed through each pair of regions within the AAL3 atlas.71 The standard implementation of the Lesion Quantification Toolkit outputs disconnection severity matrices (i.e. the number of streamlines that intersect the lesion territory). To compute a connectivity matrix, the number of preserved streamlines was estimated as the number of streamlines within the HCP-842 tractography atlas that did not intersect with the lesion territory. Graph theoretical measures quantifying the network-level impact of the lesion were derived using DSI Studio’s implementation of the Brain Connectivity Toolbox31 applied to each participant’s structural connectivity matrix where each element in the matrix corresponds to the count of preserved streamline connections between a pair of regions.In order to generate network metrics, a structural connectivity matrix was constructed for each participant by starting with the streamline trajectories included in the HCP-842 tractography atlas,  76 middle frontal gyrus, inferior frontal gyrus (IFG) , precentral gyrus, postcentral gyrus, insula, superior portion of the temporal pole, middle portion of the temporal pole, inferior temporal gyrus, middle temporal gyrus, superior temporal gyrus, supramarginal gyrus (SMG) and angular gyrus. The global, weighted network measures for the whole brain and the regional language network were used for subsequent analyses.Structural connectivity matrices and corresponding graph theoretical measures were also generated for a language sub-network comprised of the following left hemisphere regions within the AAL3 atlas and based on prior work using similar methodsThree graph theoretical measures were selected for analyses  information must travel through more steps to get from one node to another, suggesting a less functionally integrated network.31 It is associated with reduced functional connectivity,79 degree of language impairment,68 and fluency in particular32 (some studies refer to path length as ‘propagation speed’ or ‘propagation steps’).Global Efficiency: the average of each individual node’s global efficiency, which is calculated by starting with the shortest path lengths between that node and all other nodes (as for characteristic path length), taking the inverse of those path lengths, and averaging those values. Like characteristic path length, global efficiency is a measure of network integration that captures how quickly information can travel between brain regions,80 but the difference in their calculation has substantial implications. If two nodes are not connected, the path length between them is infinite and the communication efficiency is 0. When calculating the average, these values have a radically different impact . More generally, the characteristic path length is strongly influenced by long paths (infinite path length between disconnected nodes is an extreme case) but efficiency is more sensitive to short paths. This difference may be further exaggerated when these measures are calculated for stroke-damaged brains, which are more likely to have disconnected nodes or to rely on longer paths to communicate around the lesion territory. Johnson et al.77 found that higher global efficiency within the semantic network was associated with better response to naming therapy in chronic post-stroke aphasia.81 with the bifactor rotation implementation from Jennrich and Bentler.82 The behavioural measures were checked for factorability with a Kaiser–Meyer–Olkin (KMO) test; conventionally, KMO > 0.7 is considered adequate for factor analysis and KMO < 0.5 is unacceptable. A scree plot, combined with a parallel analysis and Velicer's minimum average partial (MAP) test, was used to determine the number of dimensions that should be retained.PCA was implemented using the psych package version 1.9.12,https://github.com/dorianps/LESYMAP). SCCAN LSM was run separately for each of the factors that resulted from the PCA , with sparseness optimized independently for each analysis using 8-fold CV (the number of folds was increased from the default 4-fold CV to improve stability of the results). The LSM solution was evaluated based on the correlation between predicted and observed behavioural scores. This evaluation applies to the overall LSM solution, not to individual voxels within the solution, so it is only one test and does not require correction for multiple comparisons.The SCCAN LSM analysis was conducted in R version 3.6 using the package LESYMAP version 0.0.0.9221 .Associations between connectivity measures and behavioural deficits were tested first using bivariate correlations, then multiple regression to assess simultaneous impact of multiple types of connectivity damage. Separate regressions were run for each behavioural deficit type and connectivity damage group . Several of the connectivity measures had skewed or bimodal distributions, so spearman rank correlations were used for bivariate correlations and multiple regression analyses used robust standard error estimationBivariate correlations among the behavioural measures are shown in Factor loadings from a four-factor PCA with bifactor rotation are shown in deficit). The last factor was labelled ‘Semantics’ because it had high negative loadings from the proportion of semantic errors from the PNT and high positive loadings on the percentage of correct responses from the CCT and the semantic discrimination task.The second factor had high positive loadings of lexical syntax measures that are considered critical for detecting agrammatic speech production  and moderate loadings from sentence planning measures . We will refer to this factor as ‘lexical syntax’. The third factor had high negative loadings from tests of repetition of words (PRT) and non-words (non-word repetition task) and a high positive loading from the proportion of phonological errors in picture naming, so it was named ‘Phonology’ , so it is also possible that their omission is a result of a phonological/articulatory deficit and would cluster with other measures of phonological production deficits. Neither of these turned out to be the case in the present data: the inflection index measure clustered with general measures of aphasia severity (WAB AQ), fluency (words per minute), and sentence and utterance length. Determiners are also small phonological elements that primarily serve a syntactic function, so one might expect them to pattern with inflections. Like inflection index, determiner index did strongly load on the first factor, but it also strongly loaded on the second  factor. In contrast, use of pronouns—which are short and primarily serve a structural role—loaded almost exclusively on the lexical syntax factor (with verbs and closed class words), not on the first factor or on the Phonological factor. This pattern highlights that the ‘lexical syntax’ factor appears to be specific to production of lexical elements that primarily serve a syntactic (rather than semantic) function more so than other aspects of syntax.In addition to the overall factor structure, the data-driven PCA approach yielded non-obvious observations about individual measures, such as the inflection index, determiner index and use of pronouns. Inflections are syntactic elements, and their omission is associated with agrammatism,Lesion coverage was good throughout the left middle cerebral artery (MCA) territory, particularly the dorsal speech production system structures of the frontal lobe and inferior parietal lobe . SCCAN LP < 0.001): SMG and postcentral gyrus, posterior superior temporal gyrus and middle temporal gyrus, and Heschl’s gyrus : IFG pars triangularis, precentral gyrus, and the dorsal portion of the inferior parietal lobule .Deficits on the lexical syntax factor were associated with damage in a relatively small set of fronto-parietal regions , P = 0.030], lexical syntax  and semantics  but not phonology . None of the tracts had statistically significant associations; full regression results are reported in The behavioural scores tended to be more strongly correlated with lesion volume than with connectivity measures see . TherefoEstimate = 1.424, 95% CI = , P = 0.037], Lexical syntax , Phonology  and Semantics . Language network average clustering coefficient was also a significant predictor of Phonology  and Semantics , and a marginally significant predictor of Lexical syntax .In analogous analyses of the graph theoretical measures of connectivity, the same lesion volume effects were statistically significant. In addition, language network efficiency was a significant predictor for each of the four PCA factors: Complexity [These were exploratory analyses, not driven by strong hypotheses, and comprising a relatively large number of comparisons, so these connectome disruption results should be interpreted with caution.This study examined the cognitive and neural basis of fluent speech production deficits in post-stroke aphasia, and how they relate to the effects of overall severity, semantic deficits and phonological deficits. This was done by first combining a comprehensive set of narrative speech production measures (derived from QPA) at lexical, utterance, and sentence levels, with measures of general language impairment and measures of semantic and phonological deficits. These behavioural measures were entered into a bifactor PCA, which extracts a single general factor and a series of orthogonal (uncorrelated) domain-specific factors. The bifactor PCA revealed that QPA measures of sentence/utterance length and complexity loaded primarily on the general severity factor, along with the explicit severity measure of WAB AQ. This factor accounted for 33.1% of the variance in the data. A distinct lexical syntax factor was also detected (accounting for 16.1% of the variance). This factor was comprised primarily of the usage of words that serve a structural/grammatical role rather than a semantic role  and some aspects of sentence planning . Phonology and Semantics factors had expected loadings  with virtually no contributions from any of the QPA measures, suggesting that QPA measures capture aspects of fluent speech production that are quite distinct from phonological planning and semantic processing.57 broadly agree about a lexical–morphological factor  and a sentence-level structural factor . They disagree about whether correct use of determiners, inflections and auxiliary verbs is morphological–lexical or sentential or a separate grammatical factor, and whether use of ‘well-formed’ sentences belongs with the sentence structure measures or the separate grammatical factor. There is also uncertainty about whether words per minute belongs with the lexical–morphological factor or is a separate factor reflecting speech rate or discourse productivity. The present results converge on the points of agreement , but the inclusion of severity measures (WAB AQ and naming accuracy) and prioritizing severity by using a bifactor rotation revealed that length/complexity of speech production is closely related to overall severity. Although several studies have also documented the relationship between fluency and aphasia severity,86 it has not been recognized in studies that did not include explicit measures of severity and/or tested participants in a relatively narrow range of severity.Prior studies that examined the internal structure of QPA measures88 commensurate with their primarily structural role.The ambiguous measures  also loaded strongly on this severity-related complexity factor, although words per minute, determiner index and proportion of well-formed sentences also substantively loaded on the lexical syntax factor (as did the proportion of words in sentences). This suggests that the structural complexity of aphasic speech output is strongly related to overall aphasia severity and the separable grammatical factor is largely lexical, corresponding to the correct use of words that primarily serve structural roles and add very little independent semantic content. Production of such semantically ‘light’ words may be particularly dependent on syntactic cues and vulnerable to syntactic deficits,89 and, in the context of the framework proposed by Matchin and Hickok,6 suggest that fluent production of narrative speech—as measured by length and structural complexity of utterances or sentences—is particularly reliant on temporo-parietal regions that support high-level structural planning of longer utterances and more complex sentence structures. However, there was a substantial overlap between this factor and overall aphasia severity (WAB AQ), both in the PCA results and in the SCCAN LSM results. Both the complexity factor and aphasia severity were associated with damage throughout the lesion territory, even extending dorsally beyond traditional language areas, reflecting that damage most anywhere in the left MCA territory can produce an aphasia-relevant deficit (as we have previously found48). This suggests that a broad range of different language deficits can contribute to the overall aphasia severity score and to the length and complexity of aphasic narrative speech.The lesion correlates of these deficit dimensions (factor scores) were then evaluated using SCCAN LSM, a multivariate optimization technique that more accurately identifies symptom-relevant lesion areas, including distributed networks of brain areas. Deficits on the general complexity factor were associated with damage in a fairly large portion of the lesion territory, primarily in parietal and posterior temporal regions. These regions include the dorsal speech production stream6 IFG is associated with a broad range of language processes (including syntax) and is the subject of ongoing debate. Because the lexical syntax factor was orthogonal to the complexity factor, it is unlikely that the IFG involvement observed here reflects syntactic planning, and because the factor is also orthogonal to the Phonology factor, it is unlikely that this reflects phonological–articulatory planning. A remaining possibility is that the IFG involvement reflects the recruitment of control systems to support the difficult retrieval of semantically ‘light’ words,87 which primarily serve syntactic roles and do not have strong support from semantic representations.88Deficits on the lexical syntax factor were associated with damage to a small set of fronto-parietal regions: dorsal inferior parietal lobule (dIPL), precentral gyrus, and IFG pars triangularis. Precentral gyrus and dIPL are not regions that previous studies have consistently associated with syntactic deficits or agrammatism, which are more typically associated with posterior temporal and/or inferior frontal damage.90 Even if the tract damage measures were very good, tract disconnection and behavioural deficit measures were both strongly correlated with overall lesion volume, leaving relatively little variance for an association between tract disconnection and behavioural deficit.Tract-based measures of connectivity disruption were weakly  associated with the deficit scores after controlling for overall lesion volume. In part, this may be because atlas-based estimation of tract damage is not very effective.The graph theoretical measures of connectivity produced a few suggestive patterns. First, measures derived from a left hemisphere peri-Sylvian language network were better predictors than the same measures calculated based on the connectivity of the whole brain. This may be because connectivity within the language network is particularly important for residual language function after left hemisphere stroke  or because this is the region where there was the greatest variability between participants, since this is where most of them had lesions .80 . In this case, higher efficiency scores mean that the left hemisphere language network is more functionally integrated and the results indicate better integration of the left hemisphere language network is positively associated with length and complexity of aphasic speech output, its lexical–syntactic content, and reduced phonological deficits . Surprisingly, the efficiency of the left hemisphere language network was negatively associated with semantic ability. Semantic cognition relies on a bilateral ‘hub-and-spoke’ architecture92 and the global efficiency measure tends to be less useful for larger and sparser networks,31 so these results should be interpreted with caution.Second, within the language network, global efficiency was the most consistent predictor, being significantly associated with each of the four PCA-derived deficit measures. Global network efficiency is high when the paths between nodes are relatively short and the nodes can exchange information relatively quickly and efficientlyThird, the average clustering coefficient was significantly positively associated with performance on phonological and semantic measures . This suggests that phonological and semantic processing  are supported by relatively specialized, densely connected clusters or sub-networks, as captured by the clustering coefficient measure.N = 58) that was moderate relative to other LSM studies, with most of the participants in the mild-to-moderate range of aphasia severity , though it did include some participants with more severe aphasia. Similarly, although the sample included participants across nearly the full range of fluency, most participants were in the upper half of the range , which is traditionally considered ‘fluent’ aphasia, though individuals in this range nevertheless typically have some disruptions to fluency. This severity distribution is a natural consequence of requiring at least 150 narrative words for QPA scoring—very severely impaired participants are often unable to produce that many narrative words, especially if their impairment is severely non-fluent. Both PCA and LSM are most effective when the sample covers a broad range of severity, including participants with very mild  deficits. Therefore, the present results are informative about fluency deficits in mild-to-moderate post-stroke aphasia, but it is possible that the pattern of deficits in the severe range is somewhat different.Several key limitations need to be considered when drawing conclusions from the present results. The participant sample was limited by the availability of narrative speech samples and lesion data. This resulted in a sample size  and executive function deficits were not available for inclusion in the present study, so the results do not address how the observed patterns relate to those deficits.The behavioural data were adequate for PCA according to standard metrics (KMO > 0.7), but the PCA results need to be considered in the context of which behavioural measures were included and not included (or not available). We sought to balance detailed measures of narrative speech with key anchors . The present study included fewer grammatical measures than prior PCA studies of QPA and fluency90 We found that, after controlling for overall lesion volume, tract-based connectivity disruption was weakly  associated with the deficit scores and graph theory measures of connectivity disruption produced very mixed and inconsistent results. Although unsatisfying on their own, we hope these results will encourage further development and validation of connectivity disruption measures to quantify this important aspect of brain injury.Another key limitation is that diffusion data were not available for the participants, so connectivity disruption was estimated by overlaying each participant’s lesion map on a tractography atlas derived from the HCP-842 data. This method has been widely used, but (to our knowledge) has not been validated and we have previously expressed caution about using it.In sum, the present results demonstrate that length and complexity of narrative speech production is closely related to severity, both in the behavioural sense (i.e. aphasia severity as measured by WAB AQ) and in the neural sense (a large portion of the left MCA territory is associated with reduced length and complexity of narrative speech production). In contrast, lexical aspects of syntax  appear to be separable from the severity and associated with damage in a smaller set of fronto-parietal regions, suggesting that these deficits may be related to the impaired requirement of control systems to support retrieval and correct usage of words that primarily serve structural functions rather than adding semantic content. Finally, graph theoretic measures of language network integration (efficiency) and cohesion of sub-networks (clustering coefficient) were statistically associated with the deficit scores after controlling for lesion volume, suggesting that these measures of connectivity deserve further investigation.fcac327_Supplementary_DataClick here for additional data file.


This phenomenon is thought to be one of the adaptations that prevent the progression of heart disease. Recently, the concept of using device-based neuromodulation therapies to attenuate sympathetic activity and increase parasympathetic  activity to treat cardiovascular disease, including heart failure, was developed. Although several promising preclinical and pilot clinical studies using these strategies have been conducted, the results of clinical efficacy vary. In this review, we summarize the current literature on the plasticity of cardiac sympathetic nerves and propose potential new therapeutic targets for heart disease.The heart is electrically and mechanically controlled by the autonomic nervous system, which consists of both the sympathetic and parasympathetic systems. It has been considered that the sympathetic and parasympathetic nerves regulate the cardiomyocytes’ performance independently; however, recent molecular biology approaches have provided a new concept to our understanding of the mechanisms controlling the diseased heart through the plasticity of the autonomic nervous system. Studies have found that cardiac sympathetic nerve fibers in hypertrophic ventricles strongly express an immature neuron marker and simultaneously cause deterioration of neuronal cellular function. This phenomenon was explained by the rejuvenation of cardiac sympathetic nerves. Moreover, heart failure and myocardial infarction have been shown to cause cholinergic trans-differentiation of cardiac sympathetic nerve fibers  The pathophysiology of heart failure is complex, with a wide variety of causative diseases, including hypertension, valvular heart disease, ischemic heart disease, and cardiomyopathy. The heart has abundant sympathetic innervation, and basic and clinical studies have shown that cardiac sympathetic involvement is extremely important in the pathogenesis of heart failure . Howevervia dopamine through the action of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), which are catecholamine synthases, and is stored in storage granules. Stimulation at higher levels induces exocytosis, which releases NE into the synaptic cleft. Binding of NE to β-adrenergic receptors on the myocardial cell surface produces positive potentiation and alteration effects. However, 95% of the released NE is taken up again by sympathetic nerve endings (uptake 1) and recycled. In addition, the released NE inhibits the release of NE by negative feedback via α2c receptors in sympathetic nerve endings  is synthesized from tyrosine  endings .123I-meta-iodobenzylguanidine (MIBG) deficit.In heart failure, various abnormalities also occur in sympathetic nerve endings, with NE content in the myocardium decreasing until it is depleted as sympathetic activity is continuously increased. This has been explained by increased turnover of NE at sympathetic nerve endings and a decreased ability to retain NE due to excessive release (increased spillover) and impaired reabsorption (impaired uptake 1) . FurtherConversely, there are reports that although TH expression is decreased in animal models of heart failure, there are no quantitative changes in sympathetic nerve fibers and that functional changes are the primary focus of these changes .As described above, various studies have demonstrated abnormalities of sympathetic nerve endings in heart failure. However, this pathophysiology is not fully understood as there are many inconsistencies, such as the fact that TH expression is decreased despite increased sympathetic nerve activity.In heart failure, the renin-angiotensin system is activated along with an increase in the release of endothelin (ET-1) and cytokines; this complex association of neurohumoral factor cross-talk is thought to modify the pathogenesis of heart failure along with cardiac sympathetic activation.Nerve growth factor (NGF), also known as sympathetic nerve induction factor, belongs to the neurotrophin family and is important for neural differentiation, survival, and synapse formation . ImportaFurthermore, in a rat model of right ventricular hypertrophy, it was reported that NGF protein expression was increased in conjunction with ET-1 mRNA expression in the right ventricle, and that sympathetic nerve fiber density in the right ventricle was also markedly increased , indicatTaken together, these results suggest that cardiac sympathetic nerve fiber density is strictly regulated by ET-1 expression and cardiac NGF expression under the influence of NE exposure, indicating that anatomical cardiac innervation is altered under various pathological conditions, known as “anatomical modulation”.Fetal-type genes such as beta-myosin heavy chain, skeletal muscle-type alpha-actin, ANP, and BNP are expressed in cardiomyocytes under the pathological condition of heart failure. This phenomenon is thought to be a defense mechanism by changing their isoforms. In recent years, it has become known that fetal-type genes are expressed not only in the heart but also in the nervous system as part of the body’s response to cellular damage.Previous studies have assessed embryonic gene expression in cardiac sympathetic nerves using a model of right ventricular hypertrophy and right heart failure in which monocrotaline treatment of rats induced pulmonary hypertension . AlthougIn 1974, it was reported that the addition of cultured myocardial supernatant to cultured sympathetic neurons induces differentiation of adrenergic sympathetic neurons into cholinergic neurons, i.e., switching the neurotransmitter from norepinephrine to acetylcholine in cultured neurons . This phin vitro, but the most studied representative example of cholinergic differentiation in vivo is the sweat gland . Furthermore, the viewpoint that this phenomenon acts in a bioprotective manner is a noteworthy concept that will greatly contribute to the elucidation of the pathogenesis of cardiac sympathetic nerve fiber abnormalities in heart failure. Anatomical changes in sympathetic density mediated by humoral factors after heart failure or myocardial infarction are also considered to be part of the plasticity of cardiac sympathetic innervation in pathological conditions . HoweverThe state of increased sympathetic function observed during heart failure is one of the primitive and important compensatory mechanisms. However, this compensatory mechanism leads to further myocardial injury and impaired cardiac function. Additionally, it is the source of electrophysiologically lethal arrhythmias and a poor prognostic factor. Therefore, the body must have some compensatory mechanism to maintain myocardial protective and antiarrhythmic homeostasis, which might be an adaptive mechanism using plasticity.Recently, interventional treatments in the autonomic nerves have been developed and are called neuromodulation therapies. These include baroreceptor activation therapy (BAT), spinal cord stimulation (SCS), and carotid body ablation (CBA), which use devices that block or denervate afferent nerve reflexes that increase sympathetic nerve activity, and vagal nerve stimulation (VNS) to increase parasympathetic activity, which could be direct or indirect. The promising data of these treatment methods are accumulated in preclinical and are expected to be clinically applied .The arterial baroreceptor reflex is the most powerful regulatory system of the sympathetic nerve. By applying electrical stimulation to the baroreceptor near the carotid sinus, sympathetic nerve activity is reduced from the afferent pathway to NTS, and blood pressure lowering and deceleration effects can be expected. BAT was initially developed as a treatment for angina pectoris and hypertension, but it has been reported that NT-proBNP is lowered and exercise tolerance is improved for heart failure with reduced ejection fraction . CurrentSpinal cord stimulation (SCS) is a treatment method that stimulates the spinal cord by inserting a stimulation electrode into the epidural space and was originally developed for chronic pain syndromes and refractory angina pectoris . SCS supChemoreceptor in the carotid body is central to the reflex mechanisms that primarily sense partial pressure of oxygen in arterial blood and control respiration and sympathetic nerve activity. It is known that this acceleration of the reflex mechanism induces sympathetic nerve activity in chronic heart failure. Reportedly, excision of the carotid body has the effects of lowering sympathetic nerve activity and blood pressure, improving cardiac function and prognosis in an experimental study . As for It has been reported that parasympathetic function is reduced in heart failure and myocardial infarction through changes in baroreflex sensitivity and heart rate variability . NotablyTo date, M2 has been widely known as the muscarinic receptor distributed in the myocardium, but recently, it has been reported that M3 receptors are also present in the myocardium and their expression is increased during heart failure . FurtherThe above findings indicate that cardioprotective effects can be induced by directly or indirectly enhancing parasympathetic function in response to heart failure. These changes in muscarinic receptor expression during heart failure and the above-mentioned functional changes in cardiac sympathetic nerves to cholinergic nerves may also be considered to be purposeful changes.It has been shown that activation of vagal function improves cardiac function, prevents remodeling, and improves prognosis after heart failure and myocardial infarction . In factMoreover, other preclinical VNS studies have demonstrated that VNS reduces ventricular arrhythmias and increases ventricular electrical stability in dog and pig models of myocardial infarction . InteresThese emerging pieces of evidence of the cardioprotective effects of both invasive and non-invasive VNS suggest that this may be a feasible approach for treating cardiovascular diseases in humans.Several clinical trials following a first-in-human study  of a treA recently published meta-analysis of RCTs with invasive VNS (iVNS) found that the intervention leads to a significant improvement in NYHA functional classification, quality of life, 6-min walking test distance, and NT-proBNP levels; however, they found that there was no difference in mortality . While iRecently, transcutaneous stimulation of the auricular branch of the vagus nerve (tVNS) has attracted attention as an important non-invasive alternative approach with effects comparable to those of VNS with a surgically implanted device. Previous animal studies have shown that tVNS suppressed atrial fibrillation by down-regulating both c-fos and NGF and by preventing the loss of atrial connexins (Cx40 and Cx43), an essential component of gap junctions and a key player in the formation of AF substrate, which suggests that tVNS suppresses autonomic remodeling by modulating cardiac autonomic nervous system .Human studies of tVNS for cardiovascular disease are limited. A recent study assessing the effects of neuromodulation with tVNS observed a significant improvement in global longitudinal strain, inflammatory cytokines, and quality of life in patients with heart failure with preserved ejection fraction . Alongsivia not only direct efferent projections  but also indirect vagal afferent projections . The indirect effect of tVNS on the cardiovascular system acts through the nucleus tractus solitarius (NTS), the first synaptic station of the autonomic afferent projections in the central nervous system (The hypothesis is that tVNS acts as a neuromodulation mechanism s system . The recs system  (Figure s system .Taken together, tVNS has the potential to be a beneficial, simpler, and non-invasive therapy for cardiac autonomic tone modulation that can be used to treat cardiovascular diseases and a promising substitute for iVNS.In this review, we have outlined recent concepts regarding the anatomical and functional changes of cardiac sympathetic nerves in heart failure and their relationship to the pathophysiology of heart failure. In addition, we highlighted the therapeutic potential of neuromodulation therapies, especially VNS, for cardiovascular disease. Charles Darwin once said that “It is not the strongest of the species that survives, nor the most intelligent that survives. It is the one that is most adaptable to change.”We hope that further elucidation of the underlying molecular mechanisms combined with translational work investigating the cardioprotective effects of modulation of the autonomic nervous system, including tVNS in patients, will lead to new therapeutic strategies for cardiovascular disease.HK wrote the original manuscript. HK and KF reviewed and edited the manuscript. KF acquired funding. Both contributed to the article and approved the submitted version.