A Comparison of Sanger Sequencing and Amplicon-Based Next Generation Sequencing Approaches for the Detection of HIV-1 Drug Resistance Mutations

Background: Next-generation sequencing (NGS) kits are needed to finalise the transition from Sanger sequencing to NGS in HIV-1 genotypic drug resistance testing. Materials and Methods: We compared a homemade NGS amplicon-based protocol and the AD4SEQ HIV-1 Solution v2 (AD4SEQ) NGS kit from Arrow Diagnostics for identifying resistance-associated mutations (RAMs) above the 5% threshold in 28 plasma samples where Sanger sequencing previously detected at least one RAM. Results: The samples had a median 4.8 log [IQR 4.4–5.2] HIV-1 RNA copies/mL and were mostly subtype B (61%) and CRF02_AG (14%). Homemade NGS had a lower rate of samples with low-coverage regions (2/28) compared with AD4SEQ (13/28) (p < 0.001). Homemade NGS and AD4SEQ identified additional mutations with respect to Sanger sequencing in 13/28 and 9/28 samples, respectively. However, there were two and eight cases where mutations detected by Sanger sequencing were missed by homemade NGS and AD4SEQ-SmartVir, respectively. The discrepancies between NGS and Sanger sequencing resulted in a few minor differences in drug susceptibility interpretation, mostly for NNRTIs. Conclusions: Both the NGS systems identified additional mutations with respect to Sanger sequencing, and the agreement between them was fair. However, AD4SEQ should benefit from technical adjustments allowing higher sequence coverage

A Giant Gastric Hyperplastic Polyp in Non-Familial Juvenile Polyposis of the Stomach: A Case Report

Juvenile Polyposis Syndrome (JPS) is a rare genetic disorder characterized by juvenile polyps of the gastrointestinal tract, which may also occur in people with no family history of the disorder. It usually involves the colorectum and more rarely the stomach. Juvenile polyps can vary in size from a few millimeters to over 5cm, and, although they have microscopically specific characteristics, the distinction between them and inflammatory polyps is often difficult. Here we present a case of 41 year old woman with no family history of polyposis who underwent esophagogastroduodenoscopy (EGDS) because of anemia that showed a huge and extensive gastric lesion (10 cm in diameter) and other small polyps scattered through the stomach lumen and duodenum. The histology report revealed the big lesion and the polyps were hyperplastic polyps with infiltration of inflammatory cells. Due to clinical history and EGDS findings, the patient underwent a pylorus-preserving gastrectomy, and to define a final diagnosis genetic test was performed and showed mutation in the SMAD4 gene. Thus non-familial juvenile polyposis of the stomach was diagnosed. An original feature of the current case was the rare finding of huge hyperplastic gastric polyp in a woman with rare non-familial JPS of the stomach. In fact, to our best knowledge this is the first report describing a so giant gastric polyp in JPS of the stomach in an adult woman. Furthermore, our report suggests that it is noteworthy to consider JP as differential diagnosis in patients with non-familial polyposis, anemia or hypoproteinemia and polyps histologically diagnosed as hyperplastic and performing genetic testing for confirming diagnosis and helping in the timely and appropriate management

Role of biomechanical assessment in rotator cuff tear repair: Arthroscopic vs mini-open approach

BACKGROUND Rotator cuff (RC) tears are one of the most frequent pathologies within the shoulder girdle. Hand dominance and older age are associated with RC tears. Two different surgical procedures, the mini-open (MO) and all-arthroscopic (AA) approach, represented the standard of treatment. AIM To compare the clinical and biomechanical outcomes of two surgical techniques (AA vs MO procedure) performed to address the painful shoulder syndrome with partial or total supraspinatus tendon tear. METHODS Eighty-eight participants, 50 following RC repair with AA and 38 with MO approach, were recruited in the present cross-sectional case-control study (ORTHO-SHOULDER, Prot. 0054602). All patients underwent postoperative clinical evaluation for pain (Visual analogic scale), impairment, and disability (disability of the arm, shoulder, and hand) and limitation in daily activity (Constant-Murley score). Patients\u2019 shoulder mobility was also assessed in our Laboratory of Functional Movement through a wearable inertial sensor and surface electromyography to monitor kinematics and muscle activity during the movement on the frontal (abduction/adduction) and sagittal (flexion-extension) planes. RESULTS No statistically significant differences between the two procedures were observed in either main clinical score or range of motion. A significant increase in velocity during the movement execution and a higher contribution of upper trapezius muscles were found in the AA group compared with MO patients. CONCLUSION In terms of clinical scores, our findings were in line with previous results. However, the use of technology-based assessment of shoulder mobility has revealed significant differences between the two techniques in terms of mean velocity and pattern of muscle activation

Cryopreservation of Ovarian Tissue in Pediatric Patients

Cancer treatments improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. Ovarian tissue cryopreservation allows long-term storage of primordial follicles and represents the only possibility of preserving the potential fertility in prepubertal girls. The aim of the present study is to describe our experience in ovarian tissue cryopreservation in 45 pediatric patients. The number of follicles per square millimeter of the overall section area and follicle quality were evaluated histologically. A strong negative correlation was found between age and follicular density in patients both prior to and after chemotherapy (P < 0.0001). Damage in follicular quality, that is, increased oocyte vacuolization and detachment of the oocyte from granulosa cells, was found after chemotherapy. Ovarian tissue cryopreservation, preferably performed before initiation of chemotherapy, should be offered to pediatric patients, including prepubertal girls, at risk of sterility

Changing patterns in HIV-1 non-B clade prevalence and diversity in Italy over three decades

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Bithiazole Inhibitors of Phosphatidylinositol 4-Kinase (PI4KIIIβ) as Broad-Spectrum Antivirals Blocking the Replication of SARS-CoV-2, Zika Virus, and Human Rhinoviruses

Over half a century since the description of the first antiviral drug, “old” re-emerging viruses and “new” emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated

No impact of previous NRTIs resistance in HIV positive patients switched to DTG+2NRTIs under virological control: Time of viral suppression makes the difference

The accumulation of drug-resistance mutations on combined antiretroviral regimens (ART) backbone could affect the virological efficacy of the regimen. Our aim was to assess the impact of previous drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) on the probability of virological failure (VF) in patients, under virological control, who switched to dolutegravir (DTG)+2NRTIs regimens. All HIV-1 positive drug-experienced patients who started a regimen composed by DTG+2NRTIs [abacavir/lamivudine or tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF)/emtricitabine (FTC)] in the ARCA collaborative group with HIV-RNA &lt;50 cp/mL were included in the analysis. Patients with a previous VF to integrase inhibitors were excluded. The impact of single and combined NRTIs mutations on the probability of VF (defined as 2 consecutive HIV-RNA &gt;50 copies/mL or one HIV-RNA &gt;1000 copies/mL) was assessed by Kaplan Meier curves. A multivariable Cox regression analysis was constructed to assess factors potentially related to VF. Five hundred and eighty-eight patients were included in the analysis with a median time of viral suppression before the switch of 37 months (IQR 12-78), of whom 148 (25.2%) had at least one previous NRTIs resistance mutation. In the multivariable model no association was observed between NRTIs mutations and VF. Conversely, the duration of viral suppression before switch resulted associated with a lower risk of VF (for 1 month increase, adjusted 0.98, 95%CI 0.96-0.99; p=0.024). Previous NRTIs mutations appeared to have no impact on the risk of VF in patients switched to DTG+2NRTIs, whereas a longer interval on a controlled viremia decreased significantly the risk of VF

Probing of Exosites Leads to Novel Inhibitor Scaffolds of HCV NS3/4A Proteinase

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals

Restructuring surgical training after COVID-19 pandemic: A nationwide survey on the Italian scenario on behalf of the Italian polyspecialistic young surgeons society (SPIGC)

Introduction: The COVID-19 pandemic has led to the disruption of surgical training. Lack of communication, guidelines for managing clinical activity as well as concerns for safety in the workplace appeared to be relevant issues. This study aims to investigate how surgical training has been reorganized in Italy, almost 2 years after the outbreak of COVID-19 pandemic. Materials and methods: A 16-item-electronic anonymous questionnaire was designed through SurveyMonkey© web application. This survey was composed of different sections concerning demographic characteristics and impacts of the second COVID-19 pandemic wave on surgical and research/didactic activities. Changes applied in the training programme and activities carried out were also investigated. The survey was carried out in the period between June and October 2021. Results: Four hundred and thirty responses were collected, and 399 were considered eligible to be included in the study analysis. Three hundred and thirty-five respondents continued working in Surgical Units, with a significant reduction (less than one surgical session per week) of surgical sessions in 49.6% of them. With concern to didactic and research activities, 140 residents maintained their usual activity, while 116 reported a reduction. A sub-group analysis on resident moved to COVID-19 departments showed a reduction of research activities in 35% of them. During the period considered in this survey, the surgical training program was not substantially modified for most of participants (74.6%). Conclusion: Our survey demonstrated that surgical residency programs haven't improved 2 years after the beginning of the pandemic. Further improvements are needed to guarantee completeness of surgical training, even in emergency conditions