Clinical Implications Research Articles

Objective Methods to Assess Aorto-Iliac Calcifications: A Systematic Review.

Vascular calcifications in aorto-iliac arteries are emerging as crucial risk factors for cardiovascular diseases (CVDs) with profound clinical implications. This systematic review, following PRISMA guidelines, investigated methodologies for measuring these calcifications and explored their correlation with CVDs and clinical outcomes. Out of 698 publications, 11 studies met the inclusion criteria. In total, 7 studies utilized manual methods, while 4 studies utilized automated technologies, including artificial intelligence and deep learning for image analyses. Age, systolic blood pressure, serum calcium, and lipoprotein(a) levels were found to be independent risk factors for aortic calcification. Mortality from CVDs was correlated with abdominal aorta calcification. Patients requiring reintervention after endovascular recanalization exhibited a significantly higher volume of calcification in their iliac arteries. Conclusions: This review reveals a diverse landscape of measurement methods for aorto-iliac calcifications; however, they lack a standardized reproducibility assessment. Automatic methods employing artificial intelligence appear to offer broader applicability and are less time-consuming. Assessment of calcium scoring could be routinely employed during preoperative workups for risk stratification and detailed surgical planning. Additionally, its correlation with clinical outcomes could be useful in predicting the risk of reinterventions and amputations.

A Modern Multidisciplinary Method to Characterize Natural White Spot Lesions with 2D and 3D Assessments: A Preliminary Study.

In this preliminary study, a multidisciplinary method based on high-resolution analytical techniques (such as microcomputed tomography, Raman Microspectroscopy, scanning electron microscopy, and Vickers microhardness test) was exploited to evaluate the alterations that occur in human teeth at the initial stage of the carious lesion. To this purpose, six extracted molars displaying a natural white spot lesion (WSL) were investigated. Specific morphological, structural, and chemical parameters, such as the mineral density, indentation hardness, molecular and elemental composition, and surface micromorphology were obtained on the WSL, and the results were statistically compared (t-test, p < 0.05) to those of the sound enamel on the same tooth. In the WSL, with respect to the sound area, a decrease in the mineral density and crystallinity was detected together with differences in the molecular composition and surface microstructure, such as the occurrence of micropores and irregularities. Moreover, the elemental analysis highlighted in WSL showed a statistically significant decrease in Ca and P percentages. In conclusion, this multidisciplinary approach allows us to fully characterize the area of interest, providing a deeper knowledge of these enamel lesions, which could have important clinical implications.

Large Muscle Group Movements during Sleep in Restless Leg Syndrome: Neurophysiological and Clinical Implications.

Recently, criteria have been drawn up for Large Muscle group Movements during sleep (LMM), defined as movements lasting for 3-45 seconds in adults, which are often accompanied by changes in sleep stage, arousals, and increases in heart rate. The aim of this study was to characterize LMM in RLS, in order to better evaluate their impact on the neurophysiology of the disorder and, therefore, the possible clinical implications. Consecutive, drug-free patients diagnosed with RLS and controls, aged 18 years or more, were retrospectively enrolled. Leg movement activity - short-interval (SILMS), periodic (PLMS) and isolated (ISOLMS) leg movements during sleep - and LMM were detected and scored. One-hundred patients and 67 controls were recruited. All movement measures were significantly higher in RLS. A significant positive correlation was found between LMM and ISOLMS index but not PLMS index in both groups. LMM index showed a significant negative correlation with total sleep time, sleep efficiency, and percentage of sleep stages N3 and R, as well as a significant positive correlation with number of awakenings, and percentage of sleep stages N1 and N2 only in patients with RLS. No significant correlation was found for either LMM or PLMS index and RLS severity. Different types of movements, including SILMS, ISOLMS, and LMM, play somewhat distinct roles in sleep neurophysiology in RLS. Notably, LMM, a newly recognized category of movements, demonstrate associations with sleep architecture instability and fragmentation, arousals, and awakenings, suggesting potential clinical implications.

Establishing the relationship between subjective perception and neural responses: Insights from correlation analysis and representational similarity analysis

Exploring the relationship between sensory perception and brain responses holds important theoretical and clinical implications. However, commonly used methodologies like correlation analysis performed either intra- or inter- individually often yield inconsistent results across studies, limiting their generalizability. Representational similarity analysis (RSA), a method that assesses the perception-response relationship by calculating the correlation between behavioral and neural patterns, may offer a fresh perspective to reveal novel findings. Here, we delivered a series of graded sensory stimuli of four modalities (i.e., nociceptive somatosensory, non-nociceptive somatosensory, visual, and auditory) to/near the left or right hand of 107 healthy subjects and collected their single-trial perceptual ratings and electroencephalographic (EEG) responses. We examined the relationship between sensory perception and brain responses using within- and between-subject correlation analysis and RSA, and assessed their stability across different numbers of subjects and trials. We found that within-subject and between-subject correlations yielded distinct results: within-subject correlation revealed strong and reliable correlations between perceptual ratings and most brain responses, while between-subject correlation showed weak correlations that were vulnerable to the change of subject number. In addition to verifying the correlation results, RSA revealed some novel findings, i.e., correlations between behavioral and neural patterns were observed in some additional neural responses, such as “γ-ERS” in the visual modality. RSA results were sensitive to the trial number, but not to the subject number, suggesting that consistent results could be obtained for studies with relatively small sample sizes. In conclusion, our study provides a novel perspective on establishing the relationship between behavior and brain activity, emphasizing that RSA holds promise as a method for exploring this pattern relationship in future research.

Clinical and therapeutic implications of cavernous sinus invasion in pituitary adenomas.

Invasion of the cavernous sinus by pituitary adenomas impedes complete surgical resection, compromises biochemical remission, and increases the risk of further tumor recurrence. Accurate preoperative MRI-based diagnosis or intraoperative direct inspection of cavernous sinus invasion are essential for optimal surgical planning and for tailoring postoperative therapeutic strategies, depending on whether a total resection has been achieved, or tumoral tissue has been left in surgically inaccessible locations. The molecular mechanisms underlying the invasive behavior of pituitary adenomas remain poorly understood, hindering the development of targeted therapies. Some studies have identified genes overexpressed in pituitary adenomas invading the cavernous sinus, offering insights into the acquisition of invasive behavior. Their main limitation however lies in comparing purely intrasellar specimens obtained from invasive and non-invasive adenomas. Further, precise anatomical knowledge of the medial wall of the cavernous sinus is crucial for grasping the mechanisms of invasion. Recently, alongside the standard intrasellar surgery, extended endoscopic intracavernous surgical procedures with systematic selective resection of the medial wall of the cavernous sinus have shown promising results for invasive secreting pituitary adenomas. The first- and second-generation somatostatin agonist ligands and cabergoline are used with variable efficacy to control secretory activity and/or growth of intracavernous remnants. Tumor regrowth usually requires surgical reintervention, sometimes combined with radiotherapy or radiosurgery which is applied despite their benign nature. Unraveling the molecular pathways driving invasive behavior of pituitary adenomas and their tropism to the cavernous sinuses is the key for developing efficient innovative treatment modalities that could reduce the need for repeated surgery or radiotherapy.

Addressing metallosis in knee arthroplasty: From diagnostic challenges to innovative treatments

In this editorial, we comment on the article by Toro et al published in the recent issue of World Journal of Orthopedics . This editorial review provides a comprehensive exploration of the landscape surrounding knee arthroplasty metallosis, focusing on key aspects ranging from the mechanisms influencing susceptibility to clinical implications and advanced treatment strategies. We elucidate the complex interplay of implant design, patient-specific variables, and wear-related processes contributing to metallosis. Furthermore, we seek to shed light on diagnostic challenges, the necessity of a multidisciplinary approach, and the imperative for vigilant implant surveillance. Uni-on-uni revision, as a targeted treatment modality, is discussed, highlighting its potential to address metallosis in unicompartmental knee arthroplasty (UKA). There is a need for heightened awareness among clinicians regarding the subtle presentations of metallosis, coupled with the limitations of traditional imaging techniques. Addressing metallosis requires a collaborative, multidisciplinary approach to effectively navigate the complexities associated with this complication. Furthermore, the review emphasizes the evolving paradigm of personalized care, with uni-on-uni revision emerging as a promising surgical solution. In conclusion, the editorial outlines the dynamic nature of knee arthroplasty metallosis and its multifaceted impact on clinical practice. It calls for ongoing collaboration, education, and integration of innovative solutions to enhance diagnostic accuracy, proactive management, and overall patient outcomes in the realm of UKA.

Location of infraorbital and accessory infraorbital foramina in Iranian population: a retrospective radiological study with crucial clinical implications.

The location of infraorbital foramen (IOF) and the prevalence of accessory IOF vary among different populations. It may lead to infraorbital nerve (ION) blockage during surgery. This study aimed to assess the IOF location and AIOF frequency in Iranian people. In this retrospective cross-sectional study, 500 paranasal sinus computed tomography scans of adults were examined using the INFINITT PACS system. The distance from IOF to infraorbital margin (IOM), mid-pupillary line (MPL), midsagittal line (MSL), canine eminence (CE), and skin thickness (ST) was 8.97 ± 1.79, 5.73 ± 1.84, 24.86 ± 2.23, 20.39 ± 3.47, and 10.90 ± 2.59mm, respectively. The vertical and transverse diameters of the foramen were 3.03 ± 0.65 and 3.71 ± 0.76mm, respectively. In addition, the shape of 63.5% of the foramina was oval. The prevalence of AIOF was 9%, and its most common location was superomedial to IOF. We believe that in this study, landmarks like IOM, MPL, MSL, CE and ST could help the clinicians localize IOF and improve the ION anesthesia success rate. Furthermore, the occurrence of AIOF should be considered by physicians to reduce the chance of injuries to the infraorbital neurovascular complex.

Exploring Gut Microbiome Composition and Circulating Microbial DNA Fragments in Patients with Stage II/III Colorectal Cancer: A Comprehensive Analysis.

Colorectal cancer (CRC) significantly contributes to cancer-related mortality, necessitating the exploration of prognostic factors beyond TNM staging. This study investigates the composition of the gut microbiome and microbial DNA fragments in stage II/III CRC. A cohort of 142 patients with stage II/III CRC and 91 healthy controls underwent comprehensive microbiome analysis. Fecal samples were collected for 16S rRNA sequencing, and blood samples were tested for the presence of microbial DNA fragments. De novo clustering analysis categorized individuals based on their microbial profiles. Alpha and beta diversity metrics were calculated, and taxonomic profiling was conducted. Patients with CRC exhibited distinct microbial composition compared to controls. Beta diversity analysis confirmed CRC-specific microbial profiles. Taxonomic profiling revealed unique taxonomies in the patient cohort. De novo clustering separated individuals into distinct groups, with specific microbial DNA fragment detection associated with certain patient clusters. The gut microbiota can differentiate patients with CRC from healthy individuals. Detecting microbial DNA fragments in the bloodstream may be linked to CRC prognosis. These findings suggest that the gut microbiome could serve as a prognostic factor in stage II/III CRC. Identifying specific microbial markers associated with CRC prognosis has potential clinical implications, including personalized treatment strategies and reduced healthcare costs. Further research is needed to validate these findings and uncover underlying mechanisms.

Histology of Bronchiolar Tumor Spread Through Air Spaces.

The clinical significance of lung tumor spread through air spaces (STAS) has been extensively studied, and is recognized as a unique pattern of invasion. Previous studies of STAS have focused primarily on STAS in alveolar spaces, whereas STAS in the bronchiolar spaces (bronchiolar STAS) has been described in only a few case reports only. Here, we examined 306 cases of primary lung adenocarcinoma and found that bronchiolar STAS was present in 18%. Bronchiolar STAS was associated with an inferior prognosis, more advanced stage, and higher histologic grade. No significant difference in clinicopathological factors or prognosis was observed between cases with bronchiolar STAS and those with alveolar STAS alone. Notably, bronchiolar STAS often occurred simultaneously with alveolar STAS and endobronchial spread of adenocarcinoma, particularly when bronchiolar STAS was present outside the main tumor. We also identified cases where bronchiolar STAS and endobronchial spread of adenocarcinoma occurred simultaneously in the same bronchi or bronchioles located outside the main tumor, as well as cases with bronchiolar STAS adjacent to intrapulmonary metastatic nodules. Our results highlight the significant role of bronchiolar STAS in the aerogenous spread of adenocarcinoma cells. Bronchiolar STAS can be regarded as a histologic variant of alveolar STAS. This study also supports the idea that STAS is not a tissue processing artifact, but a true biological process with clinical implications, offering histologic evidence of aerogenous spread in lung adenocarcinoma.

Passing patients’ tests and following patients’ coaching communications in psychotherapy: an empirical study

ABSTRACT According to Control-Mastery Theory, patients attend therapy with an unconscious plan to achieve adaptive goals and disprove their pathogenic beliefs. One important way patients work to disconfirm these beliefs is by testing them. Moreover, patients coach their therapists on what they need at different times during their treatment. The aim of this study is to expand the results of previous studies suggesting that therapists’ responses that support patients’ plan predict progress. We investigated whether therapists' ability to pass patients’ tests correlated with patients' improvement in the following session and whether their ability to follow patients' coaches correlated with immediate improvement. Transcriptions of 98 sessions from six brief psychodynamic psychotherapies were assessed by 12 independent raters. The patients’ plans were formulated, and tests and coaching communications were identified. The accuracy of the therapist’s responses to these tests and coaches was rated, and the impact of the therapist’s interventions on the patient’s following communication was measured. The results showed that when a therapist’s intervention passes a patient's tests, patients show signs of improvement both immediately and in the following session, and when therapists follow patients’ coaches, patients show signs of immediate improvement. The clinical implications and limitations of the findings are discussed.

The complete anatomy of the iliolumbar artery: a meta-analysis with clinical implications.

The arterial anatomy of the pelvic region is highly variable, and variations in the anatomy of the ILA may often be observed in its point of origin. The main objective of the present meta-analysis was to provide the most up-to-date and evidence-based data regarding the complete anatomy of the iliolumbar artery (ILA. It is hoped that our results may aid in reducing possible complications associated with various procedures performed in the pelvis. To perform this meta-analysis, major online medical databases - PubMed, Scopus, Embase, Web of Science, Cochrane Library, and Google Scholar - were searched through to find all studies considering the anatomy of the ILA. The pooled prevalence of the ILA originating from the Internal Iliac Artery, in the overall analysis, was found to be 93.62% (95% CI: 82.96-99.63%). Mean diameter of the ILA was found to be 2.67 mm (Standard Error = 0.19 ; Lower Limit = 2.29 ; Upper Limit = 3.05). Mean length of the ILA was established at 12.50 mm (Standard Error = 1.64 ; Lower Limit = 9.28 ; Upper limit = 15.73). The anatomy of the ILA was found to be quite constant, in contrast to what has been discussed in the literature. The said artery originated most frequently from the internal iliac artery (93.62%). Most frequently, this artery originated from the internal iliac artery (observed in approximately 93.62% of cases). Notably, the results of our current meta-analysis indicate that the average distance between the ILA's point of origin, the lower margin of the L5 vertebra, and the bifurcation site of the common iliac artery were 43.20 mm and 28.58 mm, respectively.

Exploring rare locations of hydatid disease: a retrospective case series.

Hydatid disease, endemic in Mediterranean countries, primarily affects the liver, but can manifest in diverse organs. Non-hepatic and non-pulmonary cysts often pose diagnostic challenges. This study examines patients with hydatid cysts in atypical locations. From 2013 to 2020, our center treated 250 echinococcosis patients, among whom 11 cases (4.4%) with hydatid disease in uncommon sites were retrospectively reviewed. The distribution of unusual cyst locations and their clinical implications are discussed. Diagnosing hydatid cysts in uncommon locations is a formidable challenge. Surgeons should always contemplate the prospect of an unconventional cyst location when encountering patients with cystic masses in endemic regions. Failing to consider this possibility could lead to unfavorable outcomes.

Rebound Acid Hypersecretion after Withdrawal of Long-Term Proton Pump Inhibitor (PPI) Treatment-Are PPIs Addictive?

Proton pump inhibitors (PPIs) are widely used in the long-term treatment of gastroesophageal reflux disease (GERD) and other upper gastrointestinal disorders, such as the healing of peptic ulcers and/or prophylactic treatment of peptic ulcers. PPIs are also widely used as symptomatic treatment in patients with functional dyspepsia. One of the adverse effects of the long-term use of PPI is rebound acid hypersecretion (RAHS), which can occur after the withdrawal of PPI therapy due to a compensatory increase in gastric acid production. Mechanisms of the RAHS have been well established. Studies have shown that pentagastrin-stimulated acid secretion after the discontinuation of PPIs increased significantly compared to that before treatment. In healthy volunteers treated with PPIs, the latter induced gastrointestinal symptoms in 40-50% of subjects after the discontinuation of PPI therapy but after stopping the placebo. It is important for practicing physicians to be aware and understand the underlying mechanisms and inform patients about potential RAHS before discontinuing PPIs in order to avoid continuing unnecessary PPI therapy. This is important because RAHS may lead patients to reuptake PPIs as symptoms are incorrectly thought to originate from the recurrence of underlying conditions, such as GERD. Mechanisms of RAHS have been well established; however, clinical implications and the risk factors for RAHS are not fully understood. Further research is needed to facilitate appropriate management of RAHS in the future.

Editorial Expression of Concern: miRNA signatures in childhood sarcomas and their clinical implications.

Editorial Expression of Concern: miRNA signatures in childhood sarcomas and their clinical implications.

Abstract B010: Tumor B8T expression stratifies overall survival in high risk muscle invasive urothelial carcinoma

Abstract Background and Objective: Determining novel biomarkers to identify patients that benefit and do not benefit from immune checkpoint inhibitor (ICI) therapy is critical to avoid overtreatment. Circulating tumor DNA (ctDNA) has emerged as a biomarker that associates with overall survival (OS) benefit to ICI in patients with muscle invasive urothelial carcinoma (MIUC). However, few studies have examined whether existing immune based biomarkers stratify OS within the high risk, ctDNA(+) population. Methods: To address this, we interrogated ctDNA and RNA sequencing data from the IMvigor010 dataset of patients with high risk MIUC after cystectomy who were randomized to adjuvant atezolizumab versus observation. We correlated OS with ctDNA positivity and expression of a B cell and CD8 T cell gene signature (B8T). Key Findings: In patients who were ctDNA(+), high tumor expression of a B cell gene signature (BCGS) associated with significantly higher OS when compared with patients with low tumor BCGS, regardless of whether patients were treated with atezolizumab. In ctDNA(+) patients with high tumor expression of both BCGS and CD8 T cell gene signature (CD8TGS), e.g. B8T high/high, atezolizumab did not associate with OS benefit. Conversely, in patients with B8T high/low tumors, receipt of adjuvant atezolizumab associated with significantly increased OS (p &amp;lt; 0.001), and this benefit remained significant in multivariable analyses (p = 0.047). Notably, benefit with atezolizumab occurred in patients with B8T high/low tumors who were either ctDNA(+) (p = 0.057) or ctDNA(-) (p = 0.034). Conclusions and Clinical Implications: While tumor B8T high/high expression is a prognostic biomarker for OS in ctDNA(+) patients with high risk MIUC, these results suggest B8T high/low is a predictive biomarker for benefit from ICI. Moreover, in a small subset of patients who are ctDNA(-), tumor B8T high/low associates with benefit to ICI. This supports the utility of the B8T biomarker as a prognostic identifier in high risk MIUC, its ability to identify patients who will benefit from ICI regardless of ctDNA status, and for the first time ascertains a potential cohort of patients who are ctDNA(-) yet benefit from ICI. Citation Format: Roy Elias, Adam K. Aragaki, Jean H. Hoffman-Censits, Noah M. Hahn, David J. McConkey, Burles A. Johnson III. Tumor B8T expression stratifies overall survival in high risk muscle invasive urothelial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B010.

The anatomy of the motor branches of the sciatic nerve: an anatomical study with clinical implications.

The sciatic nerve gave the motor branches supply to: biceps femoris long and short head, semitendinosus, semimembranosus and adductor magnus muscles. The anatomy of these motor branches is highly variable. The aim of this study was to estimate the anatomy and morphometry of hamstring muscles innervation. The motor branches of the sciatic nerve were dissected from both sides from 20 cadaveric specimens (9 left and 11 right) from the 11 cadavers (4 females and 7 males) at the Department of Anatomy, Jagiellonian University Medical College Cracow. The motor branches of the sciatic nerve length, distance from piriformis muscle and number of all branches that exist from tibial nerve and common fibular nerve were measured. In most cases common fibular nerve gave off one branch to the short head of biceps femoris, in most cases the proximal hamstring tendon was innervated only by the first trunk, the highest number of branches were innervating the semimembranosus and the long head of biceps femoris, the longest branches were coming to the semimembranosus and the shortest to the proximal hamstring tendon CONCLUSIONS: The present study shows that there are various innervation types of the posterior group of thigh muscles. Knowledge of possible innervation patterns could be of utmost value to operators performing surgeries on the posterior region of the thigh.

The association between tumour heterogeneity and immune evasion mechanisms in hepatocellular carcinoma and its clinical implications.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. The emergence of combination therapy, atezolizumab (anti-PDL1, immune checkpoint inhibitor) and bevacizumab (anti-VEGF) has revolutionised the management of HCC. Despite this breakthrough, the best overall response rate with first-line systemic therapyis only about 30%, owing to intra-tumoural heterogeneity, complex tumour microenvironment and the lack of predictive biomarkers. Many groups have attempted to classify HCC based on the immune microenvironment and have consistently observed better outcomes in immunologically "hot" HCC. We summarised possible mechanisms of tumour immune evasion based on the latest literature and the rationale for combination/sequential therapy to improve treatment response. Lastly, we proposed future strategies and therapies to overcome HCC immune evasion to further improve treatment outcomes of HCC.

Pterygoideus proprius muscle: stuck between the greater wing and lateral pterygoid plate.

The muscles of mastication derive from a common embryological source, and the presence of accessory muscles in the infratemporal fossa (ITF) is uncommon. Here, we present findings from postmortem dissection of the ITF revealing a unilaterally present muscle extending from the greater wing of the sphenoid to blend inferiorly with the medial and lateral pterygoid muscles before attaching to the lateral pterygoid plate. This muscle is most consistent with the pterygoideus proprius muscle initially described in 1858. Though the exact embryological origin and function of this muscle remain speculative, these topics are nonetheless worth investigating as it may provide insight regarding the ontogeny of muscles descending from the first pharyngeal arch. Additionally, presence of the pterygoideus proprius muscle may have clinical implications and impact surrounding structures such as the mandibular division of the trigeminal nerve, maxillary artery, pterygoid venous plexus, masticatory muscles, and temporomandibular joint (TMJ).

A Two-Year Observational Study to Evaluate Conversion Rates from High- and Low-Risk Patients with Amnestic Mild Cognitive Impairment to Probable Alzheimer’s Disease in a Real-World Setting

Background: Predicting conversion to probable Alzheimer&amp;s disease (AD) from amnestic mild cognitive impairment (aMCI) is difficult but important. A nomogram was developed previously for determining the risk of 3-year probable AD conversion in aMCI. Objective: To compare the probable AD conversion rates with cognitive and neurodegenerative changes for 2 years from high- and low risk aMCI groups classified using the nomogram. Methods: This prospective, multicenter, observational study was conducted in Korea. A total of patients were classified as high- or low-risk aMCI according to the nomogram and followed-up for 2 years to compare the annual conversion rate to probable AD and brain structure changes between the two groups. Results: In total, 176 (high-risk, 85; low-risk, 91) and 160 (high-risk, 77; low-risk, 83) patients completed the 1-year and 2-year follow-up, respectively. The probable AD conversion rate was significantly higher in the high-risk (Year 1, 28.9%; Year 2, 46.1%) versus low-risk group (Year 1, 0.0%; Year 2, 4.9%, both p &lt; 0.0001). Mean changes from baseline in Seoul Neuropsychological Screening Battery-Dementia Version, Clinical Dementia Rating-Sum of Box, and Korean version of the Instrumental Activities of Daily Living scores and cortical atrophy index at Years 1 and 2 were significantly greater in the high-risk group (p &lt; 0.0001). Conclusions: The high-risk aMCI group, as determined by the nomogram, had a higher conversion rate to probable AD and faster cognitive decline and neurodegeneration change than the low-risk group. These real-world results have clinical implications that help clinicians in accurately predicting patient outcomes and facilitating early decision-making. Trial Registration: ClinicalTrials.gov (NCT03448445)

In-Host Flat-like Quasispecies: Characterization Methods and Clinical Implications.

The repeated failure to treat patients chronically infected with hepatitis E (HEV) and C (HCV) viruses, despite the absence of resistance-associated substitutions (RAS), particularly in response to prolonged treatments with the mutagenic agents of HEV, suggests that quasispecies structure may play a crucial role beyond single point mutations. Quasispecies structured in a flat-like manner (referred to as flat-like) are considered to possess high average fitness, occupy a significant fraction of the functional genetic space of the virus, and exhibit a high capacity to evade specific or mutagenic treatments. In this paper, we studied HEV and HCV samples using high-depth next-generation sequencing (NGS), with indices scoring the different properties describing flat-like quasispecies. The significance of these indices was demonstrated by comparing the values obtained from these samples with those from acute infections caused by respiratory viruses (betacoronaviruses, enterovirus, respiratory syncytial viruses, and metapneumovirus). Our results revealed that flat-like quasispecies in HEV and HCV chronic infections without RAS are characterized by numerous low-frequency haplotypes with no dominant one. Surprisingly, these low-frequency haplotypes (at the nucleotide level) exhibited a high level of synonymity, resulting in much lower diversity at the phenotypic level. Currently, clinical approaches for managing flat-like quasispecies are lacking. Here, we propose methods to identifying flat-like quasispecies, which represents an essential initial step towards exploring alternative treatment protocols for viruses resistant to conventional therapies.