Consensus recommendation for prenatal, neonatal and postnatal management of congenital cytomegalovirus infection from the European congenital infection initiative (ECCI).

Congenital cytomegalovirus (cCMV) infection carries a significant burden with a 0.64% global prevalence and a 17-20% chance of serious long-term effects in children. Since the last guidelines, our understanding, particularly regarding primary maternal infections, has improved. A cCMV guidelines group was convened under the patronage of the European Society of Clinical Virology in April 2023 to refine these insights. The quality and validity of selected studies were assessed for potential biases and the GRADE framework was employed to evaluate quality of evidence across key domains. The resulting recommendations address managing cCMV, spanning prevention to postnatal care. Emphasizing early and accurate maternal diagnosis through serological tests enhances risk management and prevention strategies, including using valaciclovir to prevent vertical transmission. The guidelines also strive to refine personalized postnatal care based on risk assessments, ensuring targeted interventions for affected families.

Predictive Potential of Cmax Bioequivalence in Pilot Bioavailability/Bioequivalence Studies, through the Alternative ƒ2 Similarity Factor Method.

Pilot bioavailability/bioequivalence (BA/BE) studies are downsized trials that can be conducted prior to the definitive pivotal trial. In these trials, 12 to 18 subjects are usually enrolled, although, in principle, a sample size is not formally calculated. In a previous work, authors recommended the use of an alternative approach to the average bioequivalence methodology to evaluate pilot studies' data, using the geometric mean (Gmean) ƒ2 factor with a cut off of 35, which has shown to be an appropriate method to assess the potential bioequivalence for the maximum observed concentration (Cmax) metric under the assumptions of a true Test-to-Reference Geometric Mean Ratio (GMR) of 100% and an inter-occasion variability (IOV) in the range of 10% to 45%. In this work, the authors evaluated the proposed ƒ2 factor in comparison with the standard average bioequivalence in more extreme scenarios, using a true GMR of 90% or 111% for truly bioequivalent formulations, and 80% or 125% for truly bioinequivalent formulations, in order to better derive conclusions on the potential of this analysis method. Several scenarios of pilot BA/BE crossover studies were simulated through population pharmacokinetic modelling, accounting for different IOV levels. A redefined decision tree is proposed, suggesting a fixed sample size of 20 subjects for pilot studies in the case of intra-subject coefficient of variation (ISCV%) > 20% or unknown variability, and suggesting the assessment of study results through the average bioequivalence analysis, and additionally through Gmean ƒ2 factor method in the case of the 90% confidence interval (CI) for GMR is outside the regulatory acceptance bioequivalence interval of [80.00-125.00]%. Using this alternative approach, the certainty levels to proceed with pivotal studies, depending on Gmean ƒ2 values and variability scenarios tested (20-60% IOV), were assessed, which is expected to be helpful in terms of the decision to proceed with pivotal bioequivalence studies.

Clinical Pharmacology of Vinpocetine: Properties Revisited and Introduction of a Population Pharmacokinetic Model for Its Metabolite, Apovincaminic Acid (AVA).

This paper examines the use of vinpocetine in the context of clinical pharmacology. The main and active metabolite of vinpocetine is apovincaminic acid (AVA). Due to the scarce information in the literature on AVA pharmacokinetics, we propose a population pharmacokinetic (PopPK) model for AVA based on a study in healthy volunteers with three different formulations of vinpocetine. The suggested PopPK model (and simulations) could be helpful in ensuring the more effective and safer use of the vinpocetine in the future given the increasing range of suggested indications for its use.

Corrigendum: Antibiotic susceptibility pattern of Portuguese environmental Legionella isolates.

[This corrects the article DOI: 10.3389/fcimb.2023.1141115.].

Pooling Saliva Sample as an Effective Strategy for the Systematic CMV Screening of Newborns-A Multicentric Prospective Study.

BACKGROUND: Cytomegalovirus is the most common cause of congenital infections worldwide. Screening all newborns in the first 2 weeks of life is the only way to detect all cases of congenital infection, allowing the monitoring of children with asymptomatic infection at birth and early intervention. AIM: In this multicenter study, we aimed to evaluate the feasibility of using a saliva pool strategy for mass screening in 7 Portuguese hospitals, and to estimate the current prevalence of this congenital infection in these hospitals. METHODS: A total of 7033 newborns were screened between June 2020 and June 2022, and 704 pools of 10 saliva samples were analyzed by polymerase chain reaction (PCR). RESULTS: Of the 704 pools analyzed, 685 were negative and 19 had positive PCR results for cytomegalovirus. After individual PCR testing, 26 newborns had positive saliva results, of which 15 were confirmed by urine testing. Thus, this study's prevalence of congenital infection was 0.21% (95% confidence interval: 0.12%-0.35%). CONCLUSIONS: In this study, the pooling strategy proved to be effective for the systematic screening of newborns, although this low prevalence raises questions regarding the cost-effectiveness of implementing universal screening. However, this prevalence is probably the result of the control measures taken during the pandemic; therefore, the rates are expected to return to prepandemic values, but only a new study after the pandemic will be able to confirm this.

Kinetics of Torque Teno Virus Viral Load Is Associated with Infection and De Novo Donor Specific Antibodies in the First Year after Kidney Transplantation: A Prospective Cohort Study.

Torque teno virus (TTV) was recently identified as a potential biomarker for the degree of immunosuppression, and potentially as a predictor of rejection and infection in solid organ transplant patients. We evaluated TTV viral load in kidney transplant (KT) patients during the first year post-transplant to examine overall kinetics and their relationships with deleterious events, including episodes of infection and the formation of de novo donor-specific antibodies (DSAs). In a single-center, prospective observational cohort study, 81 KT patients were monitored at baseline, week 1, and month 1, 3, 6, 9 and 12, post-KT, and whenever required by clinical events. Kidney function, plasma TTV load, immunoglobulins and lymphocyte subpopulations were assessed at each time point. Twenty-six patients (32.1%) presented a total of 38 infection episodes post-KT. Induction immunosuppression with thymoglobulin, compared to basiliximab, was not associated with more infections (p = 0.8093). Patients with infectious events had lower T-cells (p = 0.0500), CD8+ T-cells (p = 0.0313) and B-cells (p = 0.0009) 1 month post-KT, compared to infection-free patients. Patients with infection also showed higher increases in TTV viral loads between week 1- month 1, post-KT, with TTV viral load variations >2.65 log10 cp/mL predicting the development of infectious events during the 12-month study period (p < 0.0001; sensitivity 99.73%; specificity 83.67%). Patients who developed de novo DSAs had lower TTV DNA viral loads at month 12 after KT, compared to patients who did not develop DSA (3.7 vs. 5.3 log10 cp/mL, p = 0.0023). Briefly, evaluating early TTV viremia is a promising strategy for defining infectious risk in the 1st year post-KT. The availability of standardized commercial real-time PCR assays is crucial to further validate this as an effective tool guiding immunosuppression prescription.

Diagnosing congenital cytomegalovirus infections using archived dried blood spots: A 15-year observational study, Portugal.

BACKGROUND: Cytomegalovirus (CMV) is a leading cause of congenital infections. Dried blood spots (DBS) collected in the first week of life (Guthrie cards) have been used in the diagnosis of CMV infection outside the three-week window period following birth. The present work summarizes the results of a 15-year observational study in which DBS from 1388 children were used for a late diagnosis of congenital CMV infection. METHODS: Three groups of children were studied: (i) symptomatic (with symptoms at birth or late sequelae) (N = 779); (ii) born to mothers with serological profile of primary CMV infection (N = 75); (iii) without any information (N = 534). A highly sensitive method of DNA extraction (heat-induced) from the DBS was used. CMV DNA was detected by a nested PCR. RESULTS: In total CMV DNA was detected in 7.5% (104/1388) of children. Symptomatic children showed a low rate of CMV DNA detection (6.7%) than children born to mothers with serological profile of primary CMV infection (13.3%) (p = 0.034). Sensorial hearing loss and encephalopathy were the two clinical manifestations with the highest CMV detection rate (18.3% and 11.1%, respectively). Children whose mothers had a confirmed primary infection showed a higher rate of CMV detection (35.3%) when compared with children whose mothers had a not confirmed primary infection (6.9%) (p = 0.007). CONCLUSION: The present work emphasises the importance of testing DBS in symptomatic children even a long time after symptoms onset and in children born to mothers with serologic diagnosis of maternal primary CMV infection when they miss the diagnosis during the three-week window following birth.

Antibiotic susceptibility pattern of Portuguese environmental Legionella isolates.

Introduction: Legionnaires' Disease is a pneumonia caused by Legionella spp., currently treated empirically with fluoroquinolones and macrolides. In this study, we aim to describe the antibiotic susceptibility pattern of environmental Legionella recovered in the south of Portugal. Methods: Minimal inhibitory concentration (MIC) determination of 57 Legionella isolates (10 Lp sg 1, 32, Lp sg 2-14 15 L. spp) was achieved by broth microdilution, as described by EUCAST, for azithromycin, clarithromycin, ciprofloxacin, levofloxacin, and doxycycline. Results: Fluoroquinolones were the most active antibiotic, displaying the lowest MIC values in contrast to doxycycline which had the highest. MIC90 and epidemiological cut-off (ECOFF) values were, respectively, 0.5/1 mg/L for azithromycin, 0.125/0.25 mg/L for clarithromycin, 0.064/0.125 mg/L for ciprofloxacin, 0.125/0.125 mg/L for levofloxacin and 16/32 mg/L for doxycycline. Discussion: MIC distributions were higher than reported by EUCAST for all antibiotics. Interestingly, two phenotypically resistant isolates with high-level quinolone resistance were identified. This is the first time that MIC distributions, lpeAB and tet56 genes have been investigated in Portuguese environmental isolates of Legionella.

Alternative Analysis Approaches for the Assessment of Pilot Bioavailability/Bioequivalence Studies.

Pilot bioavailability/bioequivalence (BA/BE) studies are usually conducted and analysed similarly to pivotal studies. Their analysis and interpretation of results usually rely on the application of the average bioequivalence approach. However, due to the small study size, pilot studies are inarguably more sensitive to variability. The aim of this work is to propose alternative approaches to the average bioequivalence methodology, in a way to overcome and reduce the uncertainty on the conclusions of these studies and on the potential of test formulations. Several scenarios of pilot BA/BE crossover studies were simulated through population pharmacokinetic modelling. Each simulated BA/BE trial was analysed using the average bioequivalence approach. As alternative analyses, the centrality of the test-to-reference geometric least square means ratio (GMR), bootstrap bioequivalence analysis, and arithmetic (Amean) and geometric (Gmean) mean ƒ2 factor approaches were investigated. Methods performance was measured with a confusion matrix. The Gmean ƒ2 factor using a cut-off of 35 was the most appropriate method in the simulation conditions frame, enabling to more accurately conclude the potential of test formulations, with a reduced sample size. For simplification, a decision tree is also proposed for appropriate planning of the sample size and subsequent analysis approach to be followed in pilot BA/BE trials.

Detection of JCV or BKV viruria and viremia after kidney transplantation is not associated with unfavorable outcomes.

Studies analyzing the relationship between BK polyomavirus (BKV) or JC polyomavirus (JCV) infection and kidney transplant (KT) long term clinical outcomes are scarce. Therefore, we evaluated this relationship in a single-center retrospective cohort of 288 KT patients followed for 45.4(27.5; 62.5) months. Detection of BKV viremia in two consecutive analyses led to discontinuation of antimetabolite and initiation of mammalian target of rapamycin inhibitor. Outcome data included de novo BKV and/or JCV viremia and/or viruria after KT, death-censored graft survival and patient survival. BKV viruria and viremia were detected in 42.4% and 22.2% of KT recipients, respectively. BKV viremic patients had higher urinary BKV viral loads at the onset of viruria, when compared to nonviremic patients (7 log10 vs. 4.9 log10 cp/mL, p < 0.001). JCV viruria was identified in 38.5% of KT patients; the 5.9% of KT recipients who developed JCV viremia had higher JCV urinary viral loads at the onset of viruria, when compared to non-viremic patients (5.3 vs. 3.7 log10 cp/mL, p = 0.034). No differences were found in estimated glomerular filtration rate at the end of follow up, when comparing BKV or JCV viruric or viremic patients with nonviremic patients. No association was found between JCV or BKV viruria or viremia and death/graft failure. Therefore, higher BKV urinary viral loads at the onset could serve as an early maker of over immunosuppression. JCV and BKV replication was not associated with inferior clinical outcomes in KT patients with the above-mentioned immunosuppression strategy.

Evaluation of a Proposed Approach for the Determination of the Bioequivalence Acceptance Range for Narrow Therapeutic Index Drugs in the European Union.

Bioequivalence (BE) of products containing narrow therapeutic index (NTI) drugs in the European Union is currently established by demonstrating that the 90% confidence interval for the ratio of the population geometric means of the test compared to the reference product's AUC, and in certain cases Cmax, is included within the tighter acceptance range of 90.00−111.11%. An alternative criterion, consisting of narrowed limits based on the within-subject variability of the reference product, was recently proposed. Its performance for a three-period partial replicate design was tested by simulation in terms of power to show BE, type I error (T1E) and sample size requirements. A new condition, a constraint on the test-to-reference geometric mean ratio (cGMR) to be contained within the range of 90.00−111.11%, was also tested. The probability of showing BE when the products differ more than 10% was increased, but only if the reference product's within-subject variability was moderate-to-high. The inclusion of the additional cGMR limited this. An increase in the T1E (<7%) was observed. The inclusion of the additional cGMR did not change the highest inflation of the T1E. Finally, a significant sample size reduction was observed and the inclusion of the cGMR usually did not increase the required sample size.

Under the Umbrella of Clinical Pharmacology: Inflammatory Bowel Disease, Infliximab and Adalimumab, and a Bridge to an Era of Biosimilars.

Monoclonal antibodies (MAbs) have revolutionized the treatment of many chronic inflammatory diseases, including inflammatory bowel disease (IBD). IBD is a term that comprises two quite similar, yet distinctive, disorders-Crohn's disease (CD) and ulcerative colitis (UC). Two blockbuster MAbs, infliximab (IFX) and adalimumab (ADL), transformed the pharmacological approach of treating CD and UC. However, due to the complex interplay of pharmacology and immunology, MAbs face challenges related to their immunogenicity, effectiveness, and safety. To ease the burden of IBD and other severe diseases, biosimilars have emerged as a cost-effective alternative to an originator product. According to the current knowledge, biosimilars of IFX and ADL in IBD patients are shown to be as safe and effective as their originators. The future of biosimilars, in general, is promising due to the potential of making the health care system more sustainable. However, their use is accompanied by misconceptions regarding their effectiveness and safety, as well as by controversy regarding their interchangeability. Hence, until a scientific consensus is achieved, scientific data on the long-term effectiveness and safety of biosimilars are needed.

Copper Acts Synergistically With Fluconazole in Candida glabrata by Compromising Drug Efflux, Sterol Metabolism, and Zinc Homeostasis.

The synergistic combinations of drugs are promising strategies to boost the effectiveness of current antifungals and thus prevent the emergence of resistance. In this work, we show that copper and the antifungal fluconazole act synergistically against Candida glabrata, an opportunistic pathogenic yeast intrinsically tolerant to fluconazole. Analyses of the transcriptomic profile of C. glabrata after the combination of copper and fluconazole showed that the expression of the multidrug transporter gene CDR1 was decreased, suggesting that fluconazole efflux could be affected. In agreement, we observed that copper inhibits the transactivation of Pdr1, the transcription regulator of multidrug transporters and leads to the intracellular accumulation of fluconazole. Copper also decreases the transcriptional induction of ergosterol biosynthesis (ERG) genes by fluconazole, which culminates in the accumulation of toxic sterols. Co-treatment of cells with copper and fluconazole should affect the function of proteins located in the plasma membrane, as several ultrastructural alterations, including irregular cell wall and plasma membrane and loss of cell wall integrity, were observed. Finally, we show that the combination of copper and fluconazole downregulates the expression of the gene encoding the zinc-responsive transcription regulator Zap1, which possibly, together with the membrane transporters malfunction, generates zinc depletion. Supplementation with zinc reverts the toxic effect of combining copper with fluconazole, underscoring the importance of this metal in the observed synergistic effect. Overall, this work, while unveiling the molecular basis that supports the use of copper to enhance the effectiveness of fluconazole, paves the way for the development of new metal-based antifungal strategies.

SARS-CoV-2 Antibody Seroprevalence in Patients With Cancer on Systemic Antineoplastic Treatment in the First Wave of the COVID-19 Pandemic in Portugal.

At the time of the first wave of the COVID-19 pandemic, patients with cancer were considered to be at high risk of serious illness and had a higher exposure risk since they needed frequent and nondeferrable hospital visits. Serological tests were not routinely used, and seroprevalence in this population was unknown. A single-center, cross-sectional study was developed to determine the seroprevalence of anti-SARS-CoV-2 antibodies (Abs) in patients with cancer undergoing systemic antineoplastic treatment. One hundred patients were consecutively recruited in a two-week period (6th-20th May 2020), and serum samples were tested for the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) Abs directed against both spike (S) and nucleocapsid (N) SARS-CoV-2 proteins in two distinct time points (at recruitment and 4-8 weeks later). IgG-positive results were subject to confirmation, in the same serum sample, using two distinct assays. At the time of the first study visit, no patient had a previously confirmed diagnosis of COVID-19, one reported previous contact with a COVID-19 patient, and all had a baseline SARS-CoV-2-negative RT-PCR. Two patients tested positive for SARS-CoV-2 IgG in the first study visit, which was not confirmed in either of the two confirmatory assays. Seventy-two patients were tested at the second study visit, all with negative IgG tests. IgM was persistently positive at both study visits in one patient and was positive in another patient at the second study visit, both with negative RT-PCR and serum IgG. No patient tested positive for RT-PCR within the study timeframe. No evidence of prior or acute SARS-CoV-2 infection was documented in this cohort of patients with cancer undergoing systemic treatment, and no additional exposure risk was documented compared to general population seroprevalence studies. The study was inconclusive regarding the role of SARS-CoV-2 serology in patients with cancer in the early phase of the pandemic. This study did show that, with adherence to recommended preventive measures, it was safe to maintain systemic cancer therapy.

A Proposed Approach for the Determination of the Bioequivalence Acceptance Range for Narrow Therapeutic Index Drugs in the European Union.

The current regulatory criterion for bioequivalence of narrow therapeutic index (NTI) drugs in the European Union requires that the 90% confidence interval for the ratio of the population geometric means of the test product compared with the reference for area under the plasma concentration-time curve (AUC), and in certain cases maximum plasma drug concentration (Cmax ), to be included within the tighter acceptance range of 90.00-111.11%. As a consequence, sponsors need to recruit a higher number of subjects to demonstrate bioequivalence and this may be seen as increasing the burden for the development of generics. This "one-size-fits-all" criterion is particularly questionable when the within-subject variability of the reference product is moderate to high. As an alternative, we propose a further refined statistical approach where the acceptance range is narrowed based on the within-subject variability of the reference product of the NTI drug, similar to the one used for widening the standard 80.00-125.00% acceptance range for highly variable drugs. The 80.00-125.00% acceptance range is narrowed, only if the within-subject variability is lower than 30%, down to the current NTI acceptance range of 90.00-111.11% when the within-subject variability is 13.93% or lower. Examples within the current European Medicines Agency list of NTI drugs show a considerable reduction in required sample size for drugs like tacrolimus and colchicine, where the predicted within-subject variability is 20-30%. In these cases, this approach is less sample size demanding without any expected increase in the therapeutic risks, since patients treated with reference products with moderate to high within-subject variability are frequently exposed to bioavailability differences larger than 10%.

Lower prevalence of congenital cytomegalovirus infection in Portugal: possible impact of COVID-19 lockdown?

Cytomegalovirus (CMV) is the most frequent cause of congenital infection all over the world. Its prevalence ranges from 0.2 to 2.2%. Transmission from children to their pregnant mothers is a well-known risk factor, particularly if they attend a childcare centre. This study aims to compare the prevalence of CMV congenital infection (CMV_CI) in Portugal (Lisbon) between two studies, performed respectively in 2019 and 2020. In the 2019 study, performed in two hospitals, we found a 0.67% CMV_CI prevalence, using a pool strategy previously tested with saliva samples. In the 2020 study, using the same pool approach in four hospitals (the previous and two additional), and based on 1277 samples, the prevalence was 0.078%.Conclusion: The close temporal coincidence with COVID-19 lockdown suggests that these measures may have had a significant impact on this reduction, although other explanations cannot be ruled-out. What is Known: • Cytomegalovirus is the leading cause of congenital infection. • Behavioural measures decrease cytomegalovirus seroconversion in pregnant women. What is New: • From 2019 to 2020 there was a significant reduction in the prevalence of congenital CMV infection.

Changes in Glycolytic Pathway in SARS-COV 2 Infection and Their Importance in Understanding the Severity of COVID-19.

COVID-19 is an infectious disease caused by Coronavirus 2 (SARS-CoV-2) that may lead to a severe acute respiratory syndrome. Such syndrome is thought to be related, at least in part, to a dysregulation of the immune system which involves three main components: hyperactivity of the innate immune system; decreased production of type 1 Interferons (IFN) by SARS-CoV-2-infected cells, namely respiratory epithelial cells and macrophages; and decreased numbers of both CD4+ and particularly CD8+ T cells. Herein, we describe how excessive activation of the innate immune system and the need for viral replication in several cells of the infected organism promote significant alterations in cells' energy metabolism (glucose metabolism), which may underlie the poor prognosis of the disease in severe situations. When activated, cells of the innate immune system reprogram their metabolism, and increase glucose uptake to ensure secretion of pro-inflammatory cytokines. Changes in glucose metabolism are also observed in pulmonary epithelial cells, contributing to dysregulation of cytokine synthesis and inflammation of the pulmonary epithelium. Controlling hyperglycolysis in critically ill patients may help to reduce the exaggerated production of pro-inflammatory cytokines and optimise the actions of the adaptive immune system. In this review, we suggest that the administration of non-toxic concentrations of 2-deoxy-D-glucose, the use of GLUT 1 inhibitors, of antioxidants such as vitamin C in high doses, as well as the administration of N-acetylcysteine in high doses, may be useful complementary therapeutic strategies for these patients, as suggested by some clinical trials and/ or reports. Overall, understanding changes in the glycolytic pathway associated with COVID-19 infection can help to find new forms of treatment for this disease.