Validation of a double-color ELISpot assay of IFN-γ and IL-4 production in human peripheral blood mononuclear cells.

The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest.

Characterization of Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-Antigens in Human Sera.

Nontyphoidal Salmonella (NTS) is a leading cause of morbidity and mortality caused by enteric pathogens worldwide in both children and adults, and vaccines are not yet available. The measurement of antigen-specific antibodies in the sera of vaccinated or convalescent individuals is crucial to understand the incidence of disease and the immunogenicity of vaccine candidates. A solid and standardized assay used to determine the level of specific anti-antigens IgG is therefore of paramount importance. In this work, we presented the characterization of a customized enzyme-linked immunosorbent assay (ELISA) with continuous readouts and a standardized definition of EU/mL. We assessed various performance parameters: standard curve accuracy, dilutional linearity, intermediate precision, specificity, limits of blanks, and quantification. The simplicity of the assay, its high sensitivity and specificity coupled with its low cost and the use of basic consumables and instruments without the need of high automation makes it suitable for transfer and application to different laboratories, including resource-limiting settings where the disease is endemic. This ELISA is, therefore, fit for purpose to be used for quantification of antibodies against Salmonella Typhimurium and Salmonella Enteritidis O-antigens in human samples, both for vaccine clinical trials and large sero-epidemiological studies.

Establishment and validation of a high-throughput micro-neutralization assay for respiratory syncytial virus (subtypes A and B).

The validation of a bioanalytical method allows us to determine its validity for a designated purpose and to guarantee the reliability of its analytical results. The virus neutralization assay has proved to be suitable for the detection and quantification of specific serum-neutralizing antibodies against respiratory syncytial virus subtypes A and B. Respiratory syncytial virus is a negative-sense RNA virus and is responsible for the majority of acute lower respiratory tract infections in infants and older adults worldwide. Owing to its widespread infection, the WHO considers it a target for the development of preventive vaccines. Despite the high impact of its infections, however, only one vaccine has been recently approved. The aim of this paper is to provide a detailed validation process for the microneutralization assay and to demonstrate that this method can effectively support the efficacy assessment of candidate vaccines and the definition of correlates of protection.

Flow cytometry as an integrative method for the evaluation of vaccine immunogenicity: A validation approach.

The applied bioanalytical assays used for the evaluation of human immune responses from samples collected during clinical trials must be well characterized, fully validated and properly documented to provide reliable results. Even though recommendations for the standardization of flow cytometry instrumentation and assay validation for its clinical application have been published by several organizations, definitive guidelines are not available yet. The aim of the present paper is to provide a validation approach for flow cytometry, examining parameters such as linearity, relative accuracy, repeatability, intermediate precision, range and detection limits and specificity, in order to demonstrate and document its applicability for clinical research purposes and its possible use as one of the methods for the evaluation of vaccine immunogenicity.

SARS-CoV-2 epidemiological trend before vaccination era: a seroprevalence study in Apulia, Southern Italy, in 2020.

Aim: The present study aimed at assessing the prevalence of antibodies against SARS-CoV-2 in the general population in the province of Bari (Apulia region, Southern Italy) during the year 2020. Subject and methods: In this study, 1325 serum samples collected from January to December 2020 were tested for the presence of IgM and IgG antibodies against whole-virus SARS-CoV-2 antigen by commercial ELISA. Positive samples were further tested by in-house ELISA for the detection of anti-receptor binding domain (RBD) IgM and IgG antibodies and by micro-neutralization (MN) assay for the detection of neutralizing antibody. Results: One hundred (7.55%) samples had the presence of at least one antibody class against SARS-CoV-2 by commercial ELISA, of which 88 (6.6%) showed IgG and 19 (1.4%) showed IgM antibodies. The proportion of samples with IgG antibodies increased from 1.9% in January-February to 9.6% in November-December, while no significant increase was observed for IgM. When tested by in-house ELISA and MN assay, 17.0% and 31.6% were found positive to RBD IgG and RBD IgM, respectively, while 12.0% showed neutralizing antibody. Conclusion: The proportion of samples with SARS-CoV-2 IgG antibodies increased during 2020, especially in the second half of the year, consistent with data reported by the routine epidemiological surveillance of SARS-CoV-2 cases. Despite the high number of reported cases, the seroprevalence values are relatively low, and only a small proportion of samples had neutralizing antibodies. Supplementary Information: The online version contains supplementary material available at 10.1007/s10389-023-01834-3.

The neutralizing response to SARS-CoV-2 Omicron variants BA.1 and BA.2 in COVID-19 patients and homologous and heterologous vaccinees.

The rapid replacement of Omicron BA.1 by BA.2 sublineage is very alarming, raising the question of whether BA.2 can escape the immunity acquired after BA.1 infection. We compared the neutralizing activity toward the Omicron BA.1 and BA.2 sub-lineages in five groups: COVID-19 patients; subjects who had received two doses of mRNA vaccine; subjects naturally infected with SARS-CoV-2 who had received two doses of mRNA; and subjects who had received three doses of homologous or heterologous vaccine. The results obtained highlight the importance of vaccine boosters in eliciting neutralizing antibody responses against Omicron sub-lineages, and suggest that the adenovirus vectored vaccine elicits a lower response against BA.1 than against BA.2 sub-lineage.

Antibody Avidity and Neutralizing Response against SARS-CoV-2 Omicron Variant after Infection or Vaccination.

Background: The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in SARS-CoV-2 infected patients and vaccinees. Methods: Samples were collected from 42 SARS-CoV-2 infected patients during the first pandemic wave, 50 subjects who received 2 doses of mRNA vaccine before the Omicron wave, 44 subjects who received 3 doses of mRNA vaccine, and 35 subjects who received heterologous vaccination (2 doses of adenovirus-based vaccine plus mRNA vaccine) during the Omicron wave. Samples were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results: In patients, RBD-binding IgG titers against the wild-type virus increased with time, but remained low. High neutralizing titers against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions: Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.

Causes of sudden cardiac death in young athletes and non-athletes: systematic review and meta-analysis: Sudden cardiac death in the young.

INTRODUCTION: The etiology of sudden cardiac death (SCD) in young people continues to attract much attention. This meta-analysis aimed to identify the most frequent causes of SCD in individuals aged ≤35 years, the differences between athletes and non-athletes and geographic areas. METHODS: Studies published between 01/01/1990 and 01/31/2020 and evaluating post-mortem the aetiology of SCD in young individuals (≤35 years) were included. Individuals were divided into athletes and non-athletes. Studies that did not report separate data between athletes and non-athletes were excluded. RESULTS: Thirty-four studies met the inclusion criteria, and a total population of 5,060 victims of SCD were analyzed (2,890 athletes, 2,170 non-athletes). Comparing the causes of SCD between athletes and non-athletes, non-ischemic left ventricular scar (NILVS) (5.1% vs. 1.1%, p=0.01) was more frequent in the former, while coronary artery disease (CAD) (19.6% vs. 9.1%, p=0.009), arrhythmogenic cardiomyopathy (ACM) (11.5% vs. 4.7%, p=0.03) and channelopathies (8.4% vs. 1.9%, p=0.02) were more frequent in the latter. In studies published in the last decade, hypertrophic cardiomyopathy (HCM) (p=0.002), dilated cardiomyopathy (p=0.047), and anomalous origin of coronary arteries (AOCA) (p=0.009) were more frequently the causes of SCD in athletes while aortic dissection (0.022) was the cause in non-athletes. HCM (p=0.01) and AOCA (p=0.004) were more frequently the causes of SCD in the US while ACM (p=0.001), structurally normal heart (p=0.02), and channelopathies (p=0.02) were more frequent in Europe. CONCLUSIONS: Among the causes of SCD, NILVS was the more frequent cause in athletes, while CAD, ACM and channelopathies were more frequent causes in non-athletes. The causes of SCD differ between the US and Europe.

Evaluation and correlation between SARS-CoV-2 neutralizing and binding antibodies in convalescent and vaccinated subjects.

Since the first detection of a novel Coronavirus (SARS-CoV-2) in December 2019 in Wuhan (China), it has become crucial to assess and quantize the human humoral immune response after SARS-CoV-2 natural infection and/or vaccination. Having well standardized and reliable serological assays able to accurately measure the total IgG antibodies response as well as the neutralization dynamics, play a pivotal role for the evaluation of "second" and "third" vaccines generation and in monitoring the effect in case of reinfection in the human population caused by the original strains or new SARS-CoV-2 variants. In the present study we reported that both symptomatic convalescent and vaccinated donors showed the presence of different levels of neutralizing antibodies. In addition, vaccinated subjects presented high levels of anti-S antibodies, whereas the complete absence of anti-N antibodies, whereas convalescent patients presented high levels of both anti-S and anti-N antibodies. The evaluation of the correlation between SARS-CoV-2 neutralizing and binding antibodies in convalescent and vaccinated subjects revealed that the IgG anti-S log-values were significantly higher in the vaccinated group respect to convalescent subjects. In addition, the level of binding antibodies recognizing the S protein shows a positive linear regression when compared to neutralizing titres in both the two groups evaluated.

Effect of Repeated Freeze-Thaw Cycles on Influenza Virus Antibodies.

Background: Vaccine effectiveness relies on various serological tests, whose aim is the measurement of antibody titer in serum samples collected during clinical trials before and after vaccination. Among the serological assays required by the regulatory authorities to grant influenza vaccine release there are: Hemagglutination inhibition (HAI), microneutralization (MN), and Single Radial Hemolysis (SRH). Although antibodies are regarded to be relatively stable, limited evidences on the effect of multiple freeze-thaw cycles on the stability of antibodies in frozen serum samples are available so far. In view of this, the present paper aimed to evaluate the impact of multiple freeze-thaw cycles on influenza antibody stability, performing HAI, MN and SRH assays. Methods: Ten serum samples were divided into 14 aliquots each, stored at -20 °C and taken through a total of 14 freeze-thaw cycles to assess influenza antibody stability. Each assay measurement was carried out following internal procedures based on World Health Organization (WHO) guidelines. Results: No statistically significant effect of 14 freeze-thaw cycles on antibody stability, measured through three different assays, was observed. Conclusions: Collectively, these data demonstrated that specific influenza antibody present in serum samples are stable up to 14 freeze-thaw cycles.

Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples.

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities.

Serologically-Based Evaluation of Cross-Protection Antibody Responses among Different A(H1N1) Influenza Strains.

After the influenza H1N1 pandemic of 2009, the seasonal A/Brisbane/59/2007 strain was replaced by the A/California/07/2009 strain for the influenza virus vaccine composition. After several seasons with no indications on the occurrence of antigenic drift, A/Michigan/45/2015 was chosen as the H1N1 vaccine strain for the 2017/2018 season. Since the immune response to influenza is shaped by the history of exposure to antigenically similar strains, the potential cross-protection between seasonal human influenza vaccine strains and the emerging pandemic strains was investigated. Human serum samples were tested by hemagglutination inhibition and single radial hemolysis assays against A/Brisbane/59/2007, A/California/07/2009, and A/Michigan/45/2015 strains. Strong cross-reactions between A/California/07/2009 and A/Michigan/45/2015 strains were observed in 2009/2010, most likely induced by the start of the 2009 pandemic, and the subsequent post-pandemic seasons from 2010/2011 onward when A/California/07/2009 became the predominant strain. In the 2014/2015 season, population immunity against A/California/07/2009 and A/Michigan/45/2015 strains increased again, associated with strong cross-reactions. Whereas hemagglutination inhibition assay has a higher sensitivity for detection of new seasonal drift, the single radial hemolysis assay is an excellent tool for determining the presence of pre-existing immunity, allowing a potential prediction on the booster potential of influenza vaccines against newly emerging drifted strains.

Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays.

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.

Electromechanical delay by speckle-tracking echocardiography: A novel tool to distinguish between Brugada syndrome and isolated right bundle branch block.

BACKGROUND: The electrocardiographic (ECG) definition of Brugada syndrome (BS) can be challenging because benign ECG abnormalities, such as right bundle branch block (RBBB), may mimic pathological ECG characteristics of BrS. However, although myocardial delay and deformation can be quantified by advanced imaging, it has not yet been used to differentiate between BrS and RBBB. The aim of this study was to characterize the electro-mechanical behavior of the heart of patients with type-1 BrS and isolated complete RBBB in order to differentiate these conditions. METHODS: In this two-center study, 66 subjects were analyzed by standard and speckle-tracking echocardiography (STE): 22 type-1 BrS, 24 isolated complete RBBB, and 20 healthy subjects. The participants were not treated by any drug potentially influencing myocardial conduction. RESULTS: Standard echocardiographic parameters did not differ among the groups. The greatest right ventricular (RV) mechanical dispersion was found in RBBB. Mean absolute deviations (MADs) of time-to-peak longitudinal strain calculated for each left ventricular (LV) region were greater in patients with RBBB as compared to BrS (p < .01). No differences were found between BrS and controls (p = .36). MADs in the basal segments in RBBB group were greater than MADs found in BrS group and controls (37.3 ms vs. 26.7 ms and 29.0 ms, respectively, p < .05). The greatest differences were found in the antero-septal, anterior, lateral, and infero-septal basal segments. CONCLUSIONS: Advanced echocardiographic techniques may help to differentiate between BrS and RBBB. Indeed, STE allows to identify an electro-mechanical conduction delay in RBBB patients that is not found in patients affected by type-1 BrS.

Two-point-NGS analysis of cancer genes in cell-free DNA of metastatic cancer patients.

BACKGROUND: Although the efficacy of molecularly target agents in vitro, their use in routine setting is limited mainly to the use of anti-HER2 and antiEGFR agents in vivo. Moreover, core biopsy of a single cancer site may not be representative of the whole expanding clones and cancer molecular profile at relapse may differ with respect to the primary tumor. METHODS: We assessed the status of a large panel of cancer driver genes by cell-free DNA (cfDNA) analysis in a cohort of 68 patients with 13 different solid tumors at disease progression. Whenever possible, a second cfDNA analysis was performed after a mean of 2.5 months, in order to confirm the identified clone(s) and to check the correlation with clinical evolution. RESULTS: The approach was able to identify clones plausibly involved in the disease progression mechanism in about 65% of cases. A mean of 1.4 mutated genes (range 1-3) for each tumor was found. Point mutations in TP53, PIK3CA, and KRAS and copy number variations in FGFR3 were the gene alterations more commonly observed, with a rate of 48%, 20%, 16%, and 20%, respectively. Two-points-Next-Generation Sequencing (NGS) analysis demonstrated statistically significant correlation between allele frequency variation and clinical outcome (P = .026). CONCLUSIONS: Irrespective of the primary tumor mutational burden, few mutated genes are present at disease progression. Clinical outcome is consistent with variation of allele frequency of specific clones indicating that cfDNA two-point-NGS analysis of cancer driver genes could be an efficacy tool for precision oncology.

The cerebellum improves the precision of antisaccades by a latency-duration trade-off.

The cerebellum adapts motor responses by controlling the gain of a movement, preserving its accuracy and by learning from endpoint errors. Adaptive behavior likely acts not only in the motor but also in the sensory, behavioral, and cognitive domains, thus supporting a role of cerebellum in monitoring complex brain performances. Here, we analyzed the relationship between saccade latency, duration and endpoint error of antisaccades in a group of 10 idiopathic cerebellar atrophy (ICA) patients compared to controls. The latency distribution was decomposed in a decision time and a residual time. Both groups showed a trade-off between duration and decision time, with a peak of entropy within the range of this trade-off where the information flow was maximized. In cerebellar patients, greater reductions of duration as the time of decision increased, were associated with a lower probability for a saccade to fall near the target, with a constant low entropy outside the optimal time window. We suggest a modulation of saccade duration, depending on the latency-related decision time (accumulation of sensory and motor evidences in favor of a goal-directed movement), normally adopted to perform efficient trajectories in goal-directed saccades. This process is impaired in cerebellar patients suggesting a role for the cerebellum in monitoring voluntary motor performance by controlling the movement onset until the ambiguity of planning is resolved.

A Cross-Recurrence Analysis of the Pupil Size Fluctuations in Steady Scotopic Conditions.

Pupil size fluctuations during stationary scotopic conditions may convey information about the cortical state activity at rest. An important link between neuronal network state modulation and pupil fluctuations is the cholinergic and noradrenergic neuromodulatory tone, which is active at cortical level and in the peripheral terminals of the autonomic nervous system (ANS). This work aimed at studying the low- and high-frequency coupled oscillators in the autonomic spectrum (0-0.45 Hz) which, reportedly, drive the spontaneous pupillary fluctuations. To assess the interaction between the oscillators, we focused on the patterns of their trajectories in the phase-space. Firstly, the frequency spectrum of the pupil signal was determined by empirical mode decomposition. Secondly, cross-recurrence quantification analysis was used to unfold the non-linear dynamics. The global and local patterns of recurrence of the trajectories were estimated by two parameters: determinism and entropy. An elliptic region in the entropy-determinism plane (95% prediction area) yielded health-related values of entropy and determinism. We hypothesize that the data points inside the ellipse would likely represent balanced activity in the ANS. Interestingly, the Epworth Sleepiness Scale scores scaled up along with the entropy and determinism parameters. Although other non-linear methods like Short Time Fourier Transform and wavelets are usually applied for analyzing the pupillary oscillations, they rely on strong assumptions like the stationarity of the signal or the a priori knowledge of the shape of the single basis wave. Instead, the cross-recurrence analysis of the non-linear dynamics of the pupil size oscillations is an adaptable diagnostic tool for identifying the different weight of the autonomic nervous system components in the modulation of pupil size changes at rest in non-luminance conditions.

Reference values of left atrial size and function according to age: should we redefine the normal upper limits?

Different cut-offs have been proposed for left atrial (LA) size. Furthermore, conflicting results have been reported about the influence of age on LA size and data on the impact of age on LA myocardial function are scanty. The aim of this study was to derive references values for LA size and function in healthy subjects and to evaluate the impact of age. We conducted a systematic literature search of MEDLINE database. We included only studies evaluating healthy subjects, with age ranged between 18 and 80 years. Parameters were compared among four age groups, < 30, 30-45, > 45-60, > 60 years. Three hundred twenty-six studies met the inclusion criteria and the final population consisted of 62,821 subjects. LA volume index (LAVi) did not differ among different age groups (p = 0.21). The normal upper limit of LAVi was 24 mL/m2. LA reservoir function, measured by strain, did not differ among age groups (38 ± 3%, 32-43%; p = 0.74). Left ventricular (LV) size and function were not different among groups, except LV mass index. A decrease in E/A ratio and an increase in E/e' ratio were found with advancing age (p < 0.0001 and p = 0.001, respectively). In healthy subjects the normal upper limit of LAVi was lower than that recommended and is not influenced by advancing age. Furthermore, also LA function measured by strain was not affected by age. The current reference values of LAVi should be used with caution when applied to healthy subjects.

Cardiac Magnetic Resonance Normal Reference Values of Biventricular Size and Function in Male Athlete's Heart.

OBJECTIVES: The aim of this meta-analysis was to derive normal reference values of biventricular size and function estimated by cardiac magnetic resonance (CMR) in competitive athletes. BACKGROUND: Exercise-induced enlargement of cardiac chambers is commonly observed in competitive athletes. However, ventricular dilatation is also a common phenotypic expression of life-threatening cardiomyopathies. The use of CMR for the exclusion of pathology is growing. However, normal reference values have not been established for athletes. METHODS: The authors conducted a systematic review of English-language studies in the MEDLINE, Scopus, and Cochrane databases investigating biventricular size and function by CMR in athletes. Athletes were divided into endurance, combined, and mixed groups according to the sport practiced. The potential impact of training volume was also evaluated. RESULTS: Twenty-seven studies and 983 competitive athletes were included for CMR quantification of biventricular size and function. In this review, normal reference values are presented for biventricular size and function to be applied to male competitive athletes according to the disciplines practiced. A significant impact of training volume was demonstrated for the right ventricle: athletes practicing the largest number of training hours per week were those exhibiting the greatest degree of right ventricular remodeling. Notably, biventricular function was not significantly affected by training volume. CONCLUSIONS: The present meta-analysis defines the normal limits of biventricular size and function estimated by CMR in competitive athletes. The authors suggest using these normal reference values as an alternative to standard upper limits derived from the general population when interpreting CMR images in athletes.

Influenza D Virus: Serological Evidence in the Italian Population from 2005 to 2017.

Influenza D virus is a novel influenza virus, which was first isolated from an ailing swine in 2011 and later detected in cattle, suggesting that these animals may be a primary natural reservoir. To date, few studies have been performed on human samples and there is no conclusive evidence on the ability of the virus to infect humans. The aim of this serological study was to assess the prevalence of antibodies against influenza D virus in human serum samples collected in Italy from 2005 to 2017. Serum samples were analysed by haemagglutination inhibition and virus neutralization assays. The results showed that the prevalence of antibodies against the virus increased in the human population in Italy from 2005 to 2017, with a trend characterized by a sharp increase in some years, followed by a decline in subsequent years. The virus showed the ability to infect and elicit an immune response in humans. However, prevalence peaks in humans appear to follow epidemics in animals and not to persist in the human population.