Impact of COVID-19 pandemic on outpatient visit volume in cancer patients: Results of COMETA multicenter retrospective observational study.

Objective: To evaluate the impact of the COVID-19 pandemic on first and follow-up visits for cancer outpatients. Methods: This is a multicenter retrospective observational study involving three Comprehensive Cancer Care Centers (CCCCs): IFO, including IRE and ISG in Rome, AUSL-IRCCS of Reggio Emilia, and IRCCS Giovanni Paolo II in Bari) and one oncology department in a Community Hospital (Saint'Andrea Hospital, Rome). From 1 January 2020 and 31 December 2021, we evaluated the volume of outpatient consultations (first visits and follow-up), comparing them with the pre-pandemic year (2019). Results were analyzed by quarter according to the Rt (real-time indicator used to assess the evolution of the pandemic). IFO and IRCCS Giovanni Paolo II were "COVID-free" while AUSL-IRCCS RE was a "COVID-mixed" Institute. Depending on the Rt, Sain't Andrea Hospital experienced a "swinging" organizational pathway (COVID-free/ COVID-mixed). Results: Regarding the "first appointments", in 2020 the healthcare facilities operating in the North and Center of Italy showed a downward trend. In 2021, only AUSL-IRCCS RE showed an upward trend. Regarding the "follow-up", only AUSL IRCCS RE showed a slight up-trend in 2020. In 2021, IFO showed an increasing trend, while S. Andrea Hospital showed a negative plateau. Surprisingly, IRCCS Giovanni Paolo II in Bari showed an uptrend for both first appointment and follow-ups during pandemic and late pandemic except for the fourth quarter of 2021. Conclusions: During the first pandemic wave, no significant difference was observed amongst COVID-free and COVID-mixed Institutes and between CCCCs and a Community Hospital. In 2021 ("late pandemic year"), it has been more convenient to organize COVID-mixed pathway in the CCCCs rather than to keep the Institutions COVID-free. A swinging modality in the Community Hospital did not offer positive results in term of visit volumes. Our study about the impact of COVID-19 pandemic on visit volume in cancer outpatients may help health systems to optimize the post-pandemic use of resources and improve healthcare policies.

Active screening of COVID-19-associated pulmonary aspergillosis with serum beta-glucan and endotracheal aspirates galactomannan and fungal culture.

BACKGROUND: Since February 2021 active screening of COVID-19-associated pulmonary aspergillosis (CAPA) has been implemented in our institution. OBJECTIVES: To evaluate CAPA incidence in our centre and evaluate performance of our screening protocol. METHODS: We screened once per week, collecting endotracheal aspirates for fungal culture and galactomannan (GM) and serum for 1,3-ß-D-glucan (BG). In case of positivity (GM more than 4.5, platelia assay, and/or BG >7 pg/ml, wako and/or positive fungal culture), second-level investigations were performed to pursue CAPA diagnosis according to ECMM/ISHAM criteria: bronchoalveolar lavage (BAL) fungal culture and GM, chest computed tomography (CT), serum GM. RESULTS: A total of 102 patients were screened (median age 64 years, range 39-79; 28 (27.4%) females). Twenty-two patients were diagnosed with CAPA (21%). 12 patients were positive for serum BG, 17 patients were positive for endotracheal aspirates GM and 27 patients were positive for endotracheal aspirates fungal culture. Thirty-two BALs were performed, and 26 patients underwent CT chest. Following the second level investigations 61% of the patients with positive screening tests were diagnosed with CAPA. Serum BG above 20 pg/ml or positive serum GM were always associated with typical CT chest signs of aspergillosis. Compared with 1 single positive test, having 2 positive screening test was significantly more associated with CAPA diagnosis (p = .0004). CONCLUSIONS: Active CAPA screening with serum 1,3-ß-D-glucan and endotracheal aspirates galactomannan and fungal cultures and consequent second level investigations led to high number of CAPA diagnosis. Combining more positive fungal biomarkers was more predictive of CAPA diagnosis.

Comparable cytokine release ex-vivo by whole blood from COVID-19 patients with and without non-invasive ventilation.

T cells are key players in the resolution of the infection by SARS-CoV-2. A delay in their activation can lead to severe COVID-19. The present work aimed to identify differences in cytokine release by T cells ex-vivo between COVID-19 patients in the acute phase, showing diverse disease severity. Concentrations of IFNγ, Granzyme B, IL-6, IL-10, IL-17A, IL-18, IP-10, MCP-1, and TNFα were evaluated after stimulation ex-vivo of whole blood samples with peptides from SARS-CoV-2 spike protein and a mitogen as well as without stimulation. Samples derived from hospitalized COVID-19 patients and SARS-CoV-2 vaccinated controls (CTR). Patients were classified on disease severity considering the necessity of non-invasive ventilation (NIV). Samples from patients requiring NIV revealed a similar release of cytokines compared with patients without NIV. COVID-19 patients showed higher spontaneous production of IFNγ and IP-10, lower production of MCP-1 after SARS-CoV-2 peptide stimulation and lower production of IFNγ, IL-10, IL-17A, Granzyme B, IP-10 after mitogenic stimulus compared with CTR. In conclusion, differences in T cell responses evaluated ex-vivo by a whole blood-based cytokine release assay do not appear to explain the need for non-invasive ventilation in COVID-19 patients.

Intravenous methylprednisolone pulses in hospitalised patients with severe COVID-19 pneumonia: a double-blind, randomised, placebo-controlled trial.

RATIONALE: Pulse glucocorticoid therapy is used in hyperinflammation related to coronavirus disease 2019 (COVID-19). We evaluated the efficacy and safety of pulse intravenous methylprednisolone in addition to standard treatment in COVID-19 pneumonia. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, 304 hospitalised patients with COVID-19 pneumonia were randomised to receive 1 g of methylprednisolone intravenously for three consecutive days or placebo in addition to standard dexamethasone. The primary outcome was the duration of patient hospitalisation, calculated as the time interval between randomisation and hospital discharge without the need for supplementary oxygen. The key secondary outcomes were survival free from invasive ventilation with orotracheal intubation and overall survival. RESULTS: Overall, 112 (75.4%) out of 151 patients in the pulse methylprednisolone arm and 111 (75.2%) of 150 in the placebo arm were discharged from hospital without oxygen within 30 days from randomisation. Median time to discharge was similar in both groups (15 days, 95% CI 13.0-17.0 days and 16 days, 95% CI 13.8-18.2 days, respectively; hazard ratio (HR) 0.92, 95% CI 0.71-1.20; p=0.528). No significant differences between pulse methylprednisolone and placebo arms were observed in terms of admission to intensive care unit with orotracheal intubation or death (20.0% versus 16.1%; HR 1.26, 95% CI 0.74-2.16; p=0.176) or overall mortality (10.0% versus 12.2%; HR 0.83, 95% CI 0.42-1.64; p=0.584). Serious adverse events occurred with similar frequency in the two groups. CONCLUSIONS: Methylprenisolone pulse therapy added to dexamethasone was not of benefit in patients with COVID-19 pneumonia.

Early versus delayed antiretroviral therapy based on genotypic resistance test: Results from a large retrospective cohort study.

Rapid start of antiretroviral therapy (ART) pending genotypic resistance test (GRT) has been recently proposed, but the effectiveness of this strategy is still debated. The rate of virological success (VS), defined as HIV-RNA < 50 copies/ml, with and without GRT was compared in drug-naïve individuals enrolled in the Italian ARCA cohort who started ART between 2015 and 2018. 521 individuals started ART: 397 without GRT (pre-GRT group) and 124 following GRT (post-GRT group). Overall, 398 (76%) were males and 30 (6%) were diagnosed with AIDS. In the pre-GRT group, baseline CD4+ cell counts were lower (p < 0.001), and viral load was higher (p < 0.001) than in the post-GRT group. The estimated probability of VS in pre-GRT versus post-GRT group was 72.54% (CI95 : 67.78-76.60) versus 66.94% (CI95 : 57.53-74.26) at Week 24 and 92.40% (CI95 : 89.26-94.62) versus 92.92% (CI95 : 86.35-96.33) at Week 48, respectively (p = 0.434). At Week 48, VS was less frequent among individuals with baseline CD4+ cell counts <200 versus >500 (90.33% vs. 97.33%), log viral load <5.00 versus >5.70 log10 cps/ml (97.17% vs 78.16%; p < 0.001), and those treated with protease inhibitors or non-nucleoside reverse transcriptase inhibitors versus those treated with integrase strand transfer inhibitors (p < 0.001). The rate of VS does not seem to be affected by an early ART initiation pending GRT results, but it could be influenced by the composition of the ART regimen, as well as immuno-virological parameters.

Predictive factors of clinical outcomes in patients with COVID-19 treated with tocilizumab: A monocentric retrospective analysis.

OBJECTIVE: The aim of this retrospective observational study is to analyse clinical, serological and radiological predictors of outcome in patients with COVID-19 pneumonia treated with tocilizumab, providing clinical guidance to its use in real-life. METHOD: This is a retrospective, monocentric observational cohort study. All consecutive patients hospitalized between February the 11th and April 14th 2020 for severe COVID-19 pneumonia at Reggio Emilia AUSL and treated with tocilizumab were enrolled. The patient's clinical status was recorded every day using the WHO ordinal scale for clinical improvement. Response to treatment was defined as an improvement of one point (from the status at the beginning of tocilizumab treatment) during the follow-up on this scale. Bivariate association of main patients' characteristics with outcomes was explored by descriptive statistics and Fisher or Kruskal Wallis tests (respectively for qualitative or quantitative variables). Each clinically significant predictor was checked by a loglikelihood ratio test (in univariate logistic models for each of the considered outcomes) against the null model. RESULTS: A total of 173 patients were included. Only hypertension, the use of angiotensin-converting enzyme inhibitors, PaO2/FiO2, respiratory rate and C-reactive protein were selected for the multivariate analysis. In the multivariable model, none of them was significantly associated with response. CONCLUSIONS: Evaluating a large number of clinical variables, our study did not find new predictors of outcome in COVID19 patients treated with tocilizumab. Further studies are needed to investigate the use of tocilizumab in COVID-19 and to better identify clinical phenotypes which could benefit from this treatment.

Predictors of Virological Failure Among People Living with HIV Switching from an Effective First-Line Antiretroviral Regimen.

Aim of this study was to assess the predictors of virological failure (VF) among patients living with HIV (PLWHIV) switching from an effective first-line antiretroviral therapy (ART) regimen, and to evaluate the emergence of resistance-associated mutations. All adult patients enrolled in the Antiviral Response Cohort Analysis cohort who started ART after 2010, with at least 6 months of virological suppression (VS) before ART switch and with an available genotypic resistance test (GRT) at baseline were included. Thirty-two patients out of the 607 PLWHIV included (5.3%) experienced VF after a median of 11 months from ART switch. Younger age (adjusted Hazard Ratio [aHR] 0.96, 95% confidence interval [CI] 0.92-0.99, p = .023), being male who have sex with male (aHR 0.15, 95% CI 0.03-0.69, p = .014), and longer time from VS to ART switch (aHR 0.97, 95% CI 0.95-1.00, p = .021) resulted protective toward VF, while receiving a first-line regimen containing a backbone other than ABC/3TC or TXF/FTC (aHR 3.61, 95% CI 1.00-13.1, p = .050) and a boosted protease inhibitor as anchor drug (aHR 3.34, 95% CI 1.20-9.28, p = .021) were associated with higher risk of VF. GRT at the moment of VF was available only for 13 patients (40.6%). ART switch in patients with stable control of HIV infection is a safe practice, even if particular attention should be paid in certain cases of patients switching from regimens containing low-performance backbones or protease inhibitors.

Tocilizumab or glucocorticoids treatment for patients with SARS-CoV-2 pneumonia: An observational study.

OBJECTIVE: To estimate the effect of tocilizumab or glucocorticoids in preventing death and intubation in patients hospitalized with SARS-CoV-2 pneumonia. METHODS: This was a retrospective cohort study enrolling all consecutive patients hospitalized at Reggio Emilia AUSL between February the 11th and April 14th 2020 for severe COVID-19 and treated with tocilizumab or glucocorticoids (at least 80 mg/day of methylprednisolone or equivalent for at least 3 days). The primary outcome was death within 30 days from the start of the considered therapies. The secondary outcome was a composite outcome of death and/or intubation. All patients have been followed-up until May 19th 2020, with a follow-up of at least 30 days for every patient. To reduce confounding due to potential non-comparability of the two groups, those receiving tocilizumab and those receiving glucocorticoids, a propensity score was calculated as the inverse probability weighting of receiving treatment conditional on the baseline covariates. RESULTS AND CONCLUSION: Therapy with tocilizumab alone was associated with a reduction of deaths (OR 0.49, 95% CI 0.21-1.17) and of the composite outcome death/intubation (OR 0.35, 95% CI 0.13-0.90) compared to glucocorticoids alone. Nevertheless, this result should be cautiously interpreted due to a potential prescription bias.

Tocilizumab or glucocorticoids treatment for patients with SARS-CoV-2 pneumonia: An observational study

Abstract Objective To estimate the effect of tocilizumab or glucocorticoids in preventing death and intubation in patients hospitalized with SARS-CoV-2 pneumonia. Methods This was a retrospective cohort study enrolling all consecutive patients hospitalized at Reggio Emilia AUSL between February the 11th and April 14th 2020 for severe COVID-19 and treated with tocilizumab or glucocorticoids (at least 80 mg/day of methylprednisolone or equivalent for at least 3 days). The primary outcome was death within 30 days from the start of the considered therapies. The secondary outcome was a composite outcome of death and/or intubation. All patients have been followed-up until May 19th 2020, with a follow-up of at least 30 days for every patient. To reduce confounding due to potential non-comparability of the two groups, those receiving tocilizumab and those receiving glucocorticoids, a propensity score was calculated as the inverse probability weighting of receiving treatment conditional on the baseline covariates. Results and conclusion Therapy with tocilizumab alone was associated with a reduction of deaths (OR 0.49, 95% CI 0.21-1.17) and of the composite outcome death/intubation (OR 0.35, 95% CI 0.13-0.90) compared to glucocorticoids alone. Nevertheless, this result should be cautiously interpreted due to a potential prescription bias.

High-dose glucocorticoids pulse-therapy for beta-coronaviridae pneumonia: a systematic literature review and case-series of Coronavirus disease-2019.

OBJECTIVES: The results of the RECOVERY trial identified dexamethasone as the first pharmacological therapy that reduces mortality in patients with COVID-19. The aim of this paper is to conduct a systematic literature review on safety and efficacy of pulse glucocorticoid therapy for Severe Acute Respiratory Syndrome (SARS)-CoronaVirus (CoV), Middle East Respiratory Syndrome (MERS)-CoV or SARS-CoV-2 infections and describe a case-series of COVID-19 patients treated with off-label pulse doses of methylprednisolone. METHODS: We performed a systematic literature review on safety and efficacy of pulse therapy for betacoronaviridae infections as described in the protocol registered on PROSPERO (CRD42020190183). All consecutive patients admitted to Arcispedale Santa Maria Nuova di Reggio Emilia or Guastalla Hospital with COVID-19 between March 1st and April 30th, 2020 and treated with methylprednisolone 1 gram/day for at least three days were included in the case series. A retrospective review of available computed tomography (CT) scan and chest x-ray was performed independently by two radiologists blinded to clinical data, and discordances were resolved by consensus. RESULTS: Twenty papers were included for SARS, but only two were comparative and were included in the primary endpoint analysis. Likewise, eleven papers were included for COVID-19, four of which were comparative and were considered for the primary outcome analysis. Included studies for both SARS and COVID-19 are mostly retrospective and highly heterogeneous, with lethality ranging from 0% to 100% and ICU admission rate ranging from 9% to 100%. Fourteen patients were included in our case series, 7 males and 7 females. CONCLUSIONS: No randomised controlled trial is available yet for corticosteroids pulse-therapy defined as at least ≥500mg/day methylprednisolone in patients with emerging coronavirus pneumonia. Lethality among our cohort is high (4/14), but this finding should be interpreted with caution due to the fact that in our setting pulse-steroids were used in patients not eligible for other treatments because of comorbidities or as rescue therapy. The incidence of steroid-related adverse events seems low in our cohort. The quality of the evidence on glucocorticoid pulse-therapy in SARS, MERS and COVID-19 is poor. Randomised controlled trials are greatly needed.

Thermal stability, ligand binding and allergenicity data of Mus m 1.0102 allergen and its cysteine mutants.

The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled "The allergen Mus m 1.0102: cysteine residues and molecular allergology" [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins' fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of ß-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization.

The allergen Mus m 1.0102: Cysteine residues and molecular allergology.

Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine.

Diatom Allantoin Synthase Provides Structural Insights into Natural Fusion Protein Therapeutics.

Humans have lost the ability to convert urate into the more soluble allantoin with the evolutionary inactivation of three enzymes of the uricolytic pathway. Restoration of this function through enzyme replacement therapy can treat severe hyperuricemia and Lesch-Nyhan disease. Through a genomic exploration of natural gene fusions, we found that plants and diatoms independently evolved a fusion protein (allantoin synthase) complementing two human pseudogenes. The 1.85-Å-resolution crystal structure of allantoin synthase from the diatom Phaeodactylum tricornutum provides a rationale for the domain combinations observed in the metabolic pathway, suggesting that quaternary structure is key to the evolutionary success of protein domain fusions. Polyethylene glycol (PEG) conjugation experiments indicate that a PEG-modified form of the natural fusion protein provides advantages over separate enzymes in terms of activity maintenance and manufacturing of the bioconjugate. These results suggest that the combination of different activities in a single molecular unit can simplify the production and chemical modification of recombinant proteins for multifunctional enzyme therapy.

Correction: Real-life data on potential drug-drug interactions in patients with chronic hepatitis C viral infection undergoing antiviral therapy with interferon-free DAAs in the PITER Cohort Study.

[This corrects the article DOI: 10.1371/journal.pone.0172159.].

Incidence of DAA failure and the clinical impact of retreatment in real-life patients treated in the advanced stage of liver disease: Interim evaluations from the PITER network.

BACKGROUND: Few data are available on the virological and clinical outcomes of advanced liver disease patients retreated after first-line DAA failure. AIM: To evaluate DAA failure incidence and the retreatment clinical impact in patients treated in the advanced liver disease stage. METHODS: Data on HCV genotype, liver disease severity, and first and second line DAA regimens were prospectively collected in consecutive patients who reached the 12-week post-treatment and retreatment evaluations from January 2015 to December 2016 in 23 of the PITER network centers. RESULTS: Among 3,830 patients with advanced fibrosis (F3) or cirrhosis, 139 (3.6%) failed to achieve SVR. Genotype 3, bilirubin levels >1.5mg/dl, platelet count <120,000/mm3 and the sofosbuvir+ribavirin regimen were independent predictors of failure by logistic regression analysis. The failure rate was 7.6% for patients treated with regimens that are no longer recommended or considered suboptimal (sofosbuvir+ribavirin or simeprevir+sofosbuvir±ribavirin), whereas 1.4% for regimens containing sofosbuvir combined with daclatasvir or ledipasvir or other DAAs. Of the patients who failed to achieve SVR, 72 (51.8%) were retreated with a second DAA regimen, specifically 38 (52.7%) with sofosbuvir+daclatasvir, 27 (37.5%) with sofosbuvir+ledipasvir, and 7 (9.7%) with other DAAs ±ribavirin. Among these, 69 (96%) patients achieved SVR12 and 3 (4%) failed. During a median time of 6 months (range: 5-14 months) between failure and the second DAA therapy, the Child-Pugh class worsened in 12 (16.7%) patients: from A to B in 10 patients (19.6%) and from B to C in 2 patients (10.5%), whereas it did not change in the remaining 60 patients. Following the retreatment SVR12 (median time of 6 months; range: 3-12 months), the Child-Pugh class improved in 17 (23.6%) patients: from B to A in 14 (19.4%) patients, from C to A in 1 patient (1.4%) and from C to B in 2 (2.9%) patients; it remained unchanged in 53 patients (73.6%) and worsened in 2 (2.8%) patients. Of patients who were retreated, 3 (4%) had undergone OLT before retreatment (all reached SVR12 following retreatment) and 2 (2.8%) underwent OLT after having achieved retreatment SVR12. Two (70%) of the 3 patients who failed to achieve SVR12 after retreatment, and 2 (2.8%) of the 69 patients who achieved retreatment SVR12 died from liver failure (Child-Pugh class deteriorated from B to C) or HCC complications. CONCLUSIONS: Failure rate following the first DAA regimen in patients with advanced disease is similar to or lower than that reported in clinical trials, although the majority of patients were treated with suboptimal regimens. Interim findings showed that worsening of liver function after failure, in terms of Child Pugh class deterioration, was improved by successful retreatment in about one third of retreated patients within a short follow-up period; however, in some advanced liver disease patients, clinical outcomes (Child Pugh class, HCC development, liver failure and death) were independent of viral eradication.

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

PURPOSE: Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.

Real-life data on potential drug-drug interactions in patients with chronic hepatitis C viral infection undergoing antiviral therapy with interferon-free DAAs in the PITER Cohort Study.

BACKGROUND: There are few real-life data on the potential drug-drug interactions (DDIs) between anti-HCV direct-acting antivirals (DAAs) and the comedications used. AIM: To assess the potential DDIs of DAAs in HCV-infected outpatients, according to the severity of liver disease and comedication used in a prospective multicentric study. METHODS: Data from patients in 15 clinical centers who had started a DAA regimen and were receiving comedications during March 2015 to March 2016 were prospectively evaluated. The DDIs for each regimen and comedication were assigned according to HepC Drug Interactions (www.hep-druginteractions.org). RESULTS: Of the 449 patients evaluated, 86 had mild liver disease and 363 had moderate-to-severe disease. The use of a single comedication was more frequent among patients with mild liver disease (p = 0.03), whereas utilization of more than three drugs among those with moderate-to-severe disease (p = 0.05). Of the 142 comedications used in 86 patients with mild disease, 27 (20%) may require dose adjustment/closer monitoring, none was contraindicated. Of the 322 comedications used in 363 patients with moderate-to-severe liver disease, 82 (25%) were classified with potential DDIs that required only monitoring and dose adjustments; 10 (3%) were contraindicated in severe liver disease. In patients with mild liver disease 30% (26/86) used at least one drug with a potential DDI whereas of the 363 patients with moderate-to-severe liver disease, 161 (44%) were at risk for one or more DDI. CONCLUSIONS: Based on these results, we can estimate that 30-44% of patients undergoing DAA and taking comedications are at risk of a clinically significant DDI. This data indicates the need for increased awareness of potential DDI during DAA therapy, especially in patients with moderate-to-severe liver disease. For several drugs, the recommendation related to the DDI changes from "dose adjustment/closer monitoring", in mild to moderate liver disease, to "the use is contraindicated" in severe liver disease.

Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids.

Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 µM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations.

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2.

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A(2) (sPLA(2)s). TbSP1, the sPLA(2) primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A(2), whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA(2) overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.