Glucose-6-phosphate dehydrogenase (G6PD) deficiency, caused by pathogenic variants in the G6PD gene, is the most common X-linked enzymopathy in the world. The clinical manifestations range from drug-induced hemolytic anemia to neonatal hyperbilirubinemia and chronic hemolysis. The G6PD status needs to be accurately assessed in several clinical settings, including drug prescription, assisted reproductive technologies, and the diagnostic workflow of hemolytic anemia. In this study, the contribution of molecular analysis in the diagnostic workflow of patients with suspected G6PD deficiency was retrospectively evaluated over one year. A total of 340 samples were analyzed for the enzymatic activity of G6PD, including 28 organ donors, 62 pediatric patients, 157 hospitalized patients, and 93 referred from the Genetics, Internal Medicine, Gastroenterology, and Hematology units. Molecular analysis was performed for 52 cases (17 males and 35 females): 37 with reduced enzymatic activity, five with inconclusive results due to recent blood transfusions, and 10 with specific clinical indications. Through molecular testing, eight previously reported and five novel disease-causing variants (c.769 C > T, p.(Arg257Gly); c.488G > T, p.(Gly163Val); c.1124 A > G, p.(Asn375Gly); c.1152G > C p.(Gln384His); c.582 C > G, p.(Asp194Glu)) were identified in 36 patients, along with three common polymorphic variants in 11 other patients. No variants were identified in five patients, consistent with borderline or difficult-to-interpret activity results. Overall, lower G6PD activity was found in 66 of 340 samples (19.4

Acute intermittent porphyria (AIP) is a rare metabolic disorder characterized by acute attacks often triggered by porphyrinogenic drugs and a low-glucose diet. According to recent findings, chronic symptoms persist in AIP patients. To avoid the symptoms, patients often adopt preventive strategies such as increasing glucose intake, suggesting that nutrition is a crucial aspect of disease management. Given the strong connection between AIP and glucose, we assessed anthropometric data, biochemical data and nutritional evaluation, in 16 AIP females and hypothesized that an increase in glucose consumption may lead to an imbalance in nutrition and metabolism. The results indicated that 14 out of 16 patients consumed high levels of simple sugars and saturated fatty acids (SFA), leading to overweight conditions (BMI > 25) in 50

In patients with respiratory diseases, a panel of markers is often used to assess disease severity and progression. Here we test whether the serum lipid signature may surge as a reliable alternative marker to monitor systemic hypoxia, a frequent unfavourable outcome in acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary diseases (COPD). We recruited 9 healthy controls, 10 COPD patients, and 10 ARDS patients. Various markers related to inflammation, redox imbalance, and iron handling were measured alongside lipid profiles obtained through untargeted lipidomic analysis. The results show that serum lipids were moderately lower in COPD patients and significantly reduced in ARDS patients compared to the controls. Six lipid classes (cholesteryl esters, coenzyme Q, phosphatidylinositol, sterols, hexosylceramides, and phosphatidylethanolamine) exhibited significant changes (ANOVA p < 0.05) and correlated with the Horowitz index (P/F), suggesting their potential as markers of hypoxia severity. While conventional markers also correlated with P/F, the lipid signature was more specific and reliable. This study highlights that hypoxia in pulmonary diseases depresses circulating lipids, with certain lipid classes offering more precise predictions of hypoxia severity. Expanding this research to larger populations could support the lipid signature as a clinical tool.

Plasma non-transferrin-bound iron (NTBI) comprises multiple subspecies, classified by their composition, chemical reactivity, and susceptibility to chelation. The redox-active and chelatable fraction of NTBI is referred to as labile plasma iron (LPI). The pathophysiological significance of NTBI and LPI lies in their ability to enter cells via alternative transport pathways that are not regulated by the transferrin receptor system or by cellular iron levels. Several mechanisms have been proposed for their cellular entry, including the hijacking of divalent metal transporters and passive diffusion. This unregulated uptake can lead to iron accumulation in vulnerable tissues such as the liver and the heart. NTBI and LPI bypassing normal cellular control mechanisms can rapidly exceed the cell’s capacity to safely store excess iron, leading to toxicity. Both NTBI and LPI contribute to oxidative stress by participating in free-radical-generating reactions. However, LPI concentration in the bloodstream may be differentially affected by the mode and extent of iron overload, the presence of residual serum iron-binding activity, and the antioxidant capacity of individual sera. In summary, both NTBI and LPI contribute to iron-mediated toxicity but differ in terms of reactivity, availability, and pathogenic potential depending on the pathophysiological conditions that influence the degree of toxicity.

Background/Objectives: Anemia during acute inflammation is not well described in the literature. We aimed to study whether patients develop a transient hemoglobin decrease during an acute attack of recurrent pericarditis. Methods: We retrospectively analyzed patients with recurrent pericarditis. The primary endpoint was the difference in hemoglobin levels during an acute attack and in the following remission. As secondary endpoints, we correlated this variation with laboratory and clinical features; we also evaluated the available baseline hemoglobin values. Results: Sixty-two patients, including thirty females (48.4%), with a median age of 39 years, were observed during an acute attack and remission. The attack indexed was the first in 21 patients and the second or the third in 41, with pre-attack hemoglobin levels available for the latter group. Median hemoglobin levels (IQR) were 13.8 (12.8-15.1) g/dL at baseline, 12.0 (11.2-13.4) during attacks and 13.6 (13.1-14.0) during remission (p < 0.001). The median hemoglobin reduction between an acute attack and remission was 1.4 g/dL. Their mean corpuscular volume remained in the normal range. Hb reduction significantly correlated with C-reactive protein (CRP) elevation, neutrophilia and the neutrophil-to-lymphocyte ratio, but not serosal involvement. Only CRP elevation remained associated with the variation of Hb in a multivariate analysis (p = 0.007). Conclusions: This study is a proof of concept: hemoglobin levels may decline rapidly during acute inflammation in correlation with CRP elevation, with transient normocytic anemia, followed by a rapid rebound. In this regard, idiopathic pericarditis may represent a pathogenetic model of this type of anemia.

Anemia and hyperferritinemia are frequent findings at diagnosis of Gaucher disease (GD). Macrophage-independent dyserythropoiesis and abnormal iron metabolism have been shown. We evaluated hematological and iron status at diagnosis (T0) and the effect of enzyme replacement therapy (ERT) on erythropoiesis and iron utilization over 5-year follow-up in type 1 GD patients and in an ex vivo model of erythropoiesis from CD34 + peripheral blood cells. At T0, 41

Introduction & AimHDV exploits HBV surface protein (HBsAg) for releasing of progeny and entry into hepatocytes. HBsAg consists of three different proteins: Large-HBsAg (L-HBs), Middle-HBsAg (M-HBs) and small-HBsAg (S-HBs). L-HBs is predominantly present in virions and mediates binding to NTCP receptor. Here, we investigate the kinetics of HBs-forms under bulevirtide-treatment (BLV).Method28 consecutive patients with HDV-related compensated cirrhosis starting BLV-monotherapy were enrolled in this retrospective/longitudinal study, all under effective NUC-treatment at entry. L-HBs, M-HBs and S-HBs were quantified by ad-hoc ELISAs in baseline and week 48 (W48) samples (Beacle). HDV-RNA was quantified by Robogene. Virological response was defined as HDV-RNA undetectable or >2log decline compared to baseline, biochemical response as ALT normalization.ResultsAt baseline, median(IQR) age was 53(40-63) years, liver stiffness 19.4(15.4-32.9) kPa, ALT 106(77-147) U/l, serum HDV-RNA 5.4(4.4-6.0) logIU/ml and HBsAg levels 3.7(3.4-4.9) logIU/ml. Pre-treatment median(IQR) levels of S-HBs, M-HBs and L-HBs were 3999(1482-7184), 1032(305-2222) and 6(2-14) ng/ml, respectively. At W48, serum HDV-RNA declined by 3.1(1.8-3.7) logIU/ml while virological and biochemical responses were observed in 71% and 75% of patients, respectively. S-HBs, M-HBs and L-HBs decreased of >10% respect to baseline in 57%, 54% and 39% of patients with a median (IQR) decline of 1095(839-2403), 145(39-350) and 10(4-15) ng/ml, respectively.HDV-RNA<5logIU/ml at baseline was associated with HDV-RNA<100IU/ml at W48 (75% with vs 25% without, P=0.02). A similar correlation was observed for L-HBs<9 ng/ml at baseline (62.5% with vs 25% without, P=0.05). Notably, the combination of pre-treatment L-HBs<9ng/ml + HDV-RNA<5logIU/ml was the best predictor for achieving HDV-RNA<100IU/ml (PPV:87.5 % and NPV:70%; P=0.01). This combination showed also the best diagnostic accuracy for predicting HDV-RNA<100IU/ml plus ALT normalization (PPV:75%; NPV:75%; P=0.03).ConclusionQuantification of L-HBs along with serum HDV-RNA may reflect the burden of circulating infectious virions, possibly providing a new tool to identify patients more likely to respond to BLV-monotherapy.

Background In Italy, HDV-prevalence and its fluctuations over time are controversial while an extensive characterization of HDV-infected patients is missing. Aim To assess HDV-seroprevalence in a large cohort of HBsAg-positive subjects, followed in Central Italy over time, and the epidemiological/virological characteristics of HDV-infected patients. Methods 1,579 consecutive and well-characterized HBsAg-positive patients, firstly referred to Tor Vergata-University-Hospital from 2005-2022, were included. HDV-RNA was quantified by a commercial assay (LLOQ:100copies/ml) and HDV sub-genotypes were defined by phylogenetic-analysis. Results Among 1,579 HBsAg-positive patients, 45.3% (715/1579) received HDV-screening with an increasing temporal-trend: 17.1% (2005-2010), 43.2% (2011-2015), 56.5% (2016-2019), 75.8% (2020-2022). The lack of HDV-screening significantly correlated with normal ALT (OR[95%CI]:1.69[1.28-2.22], P<0.001) and being Italian (OR[95%CI]:1.4[1.12-184], P=0.005), while no factors were identified in 2020-2022. Overall, this suggests a higher awareness towards HDV-screening in all HBsAg+ in recent years. 13.4% (96/715) of HDV-screened patients resulted anti-HDV+ with a stable temporal trend: 10.7% (2005-2010), 15.6% (2011-2015), 10.8% (2016-2019), 10% (2020-2022). Among them, 80.5% had detectable HDV-RNA (median[IQR]log:4.6[3.6-5.6]copies/ml) with altered ALT in 89.3% (median[IQR]:92[62-177]U/L). Anti-HDV positivity was higher in patients from Eastern Europe than from Italy (23.6% versus 12.9%, P=0.002). Notably, anti-HDV+ patients from Eastern Europe were younger (44[37-54] versus 53[47-62]years, P<0.001) with higher HDV-RNA (4.8[3.6-5.8] versus 3.9[1.4-4.9]copies/ml, P=0.016) and HBsAg (9,461[4,159-24,532] versus 4,447[737-13,336]IU/ml), P=0.032), indicating more pronounced HDV-replication. Phylogenetic-analysis revealed the circulation of HDV sub-genotype 1a (25.9%), 1b (33.4%), 1c (25.9) and 1d (14.8%). Notably, sub-genotype 1a and 1c correlated with 3xULN ALT compared to 1b and 1d (75%% versus 27.3%, P=0.039). Conclusions The awareness to request HDV-screening is increasing over time even if some gaps remain to achieve HDV-screening in all HBsAg-positive patients. Immigration from Eastern Europe contributes to the circulation of HDV-strains with enhanced replication. The detection of different sub-genotypes, triggering variable inflammatory stimuli, supports the need to expand HDV molecular characterization. In Italy, HDV-prevalence and its fluctuations over time are controversial while an extensive characterization of HDV-infected patients is missing. To assess HDV-seroprevalence in a large cohort of HBsAg-positive subjects, followed in Central Italy over time, and the epidemiological/virological characteristics of HDV-infected patients. 1,579 consecutive and well-characterized HBsAg-positive patients, firstly referred to Tor Vergata-University-Hospital from 2005-2022, were included. HDV-RNA was quantified by a commercial assay (LLOQ:100copies/ml) and HDV sub-genotypes were defined by phylogenetic-analysis. Among 1,579 HBsAg-positive patients, 45.3% (715/1579) received HDV-screening with an increasing temporal-trend: 17.1% (2005-2010), 43.2% (2011-2015), 56.5% (2016-2019), 75.8% (2020-2022). The lack of HDV-screening significantly correlated with normal ALT (OR[95%CI]:1.69[1.28-2.22], P<0.001) and being Italian (OR[95%CI]:1.4[1.12-184], P=0.005), while no factors were identified in 2020-2022. Overall, this suggests a higher awareness towards HDV-screening in all HBsAg+ in recent years. 13.4% (96/715) of HDV-screened patients resulted anti-HDV+ with a stable temporal trend: 10.7% (2005-2010), 15.6% (2011-2015), 10.8% (2016-2019), 10% (2020-2022). Among them, 80.5% had detectable HDV-RNA (median[IQR]log:4.6[3.6-5.6]copies/ml) with altered ALT in 89.3% (median[IQR]:92[62-177]U/L). Anti-HDV positivity was higher in patients from Eastern Europe than from Italy (23.6% versus 12.9%, P=0.002). Notably, anti-HDV+ patients from Eastern Europe were younger (44[37-54] versus 53[47-62]years, P<0.001) with higher HDV-RNA (4.8[3.6-5.8] versus 3.9[1.4-4.9]copies/ml, P=0.016) and HBsAg (9,461[4,159-24,532] versus 4,447[737-13,336]IU/ml), P=0.032), indicating more pronounced HDV-replication. Phylogenetic-analysis revealed the circulation of HDV sub-genotype 1a (25.9%), 1b (33.4%), 1c (25.9) and 1d (14.8%). Notably, sub-genotype 1a and 1c correlated with 3xULN ALT compared to 1b and 1d (75%% versus 27.3%, P=0.039). The awareness to request HDV-screening is increasing over time even if some gaps remain to achieve HDV-screening in all HBsAg-positive patients. Immigration from Eastern Europe contributes to the circulation of HDV-strains with enhanced replication. The detection of different sub-genotypes, triggering variable inflammatory stimuli, supports the need to expand HDV molecular characterization.

ObjectivesGlucose-6-phosphate dehydrogenase (G6PD) deficiency adds a burden on patients and their families in Egypt and Middle East due to lifelong diet restriction. Thus, the current study aimed to investigate the challenge with non-fava beans (dicotyledons) diet on the development of hemolysis in G6PD-deficient patients as well as making a genotype-phenotype correlation from two centers.Patients and methodsA 1-year interventional study was registered in the Clinical Trials Government (NCT02498340), including 108 patients subjected to quantitative analyses for enzymatic activity and molecular typing of G6PD enzyme using a PCR-amplification refractory mutation system technique. Dietetic challenge included ingestion of non-fava beans diet taken in small amount (10-20 g/day for 3 successive days/week) weekly for 1 year with clinical and laboratory follow-up of essential markers of hemolysis.ResultsMediterranean mutation was the commonest type (53.7%) and with significantly more patients with severe enzyme deficiency in this mutation, followed by African mutation (16.7%), while Cairo mutation was expressed in 13.9%. All studied genotypes were comparable regarding their clinical presentations. After diet challenge, none of the patients with identified genotypes showed significant changes in their G6PD, mean hemoglobin, indirect bilirubin levels, and reticulocytic count (%) compared with baseline.ConclusionG6PD Mediterranean mutation is the commonest mutations causing G6PD deficiency in Egypt. G6PD-deficient children might tolerate diet that contains a small amount of non-fava beans for 1 year without inducing hemolysis, so its restriction in G6PD-deficient patients should be reconsidered.

No published study has investigated the mitochondrial count in patients suffering from acute intermittent porphyria (AIP). In order to determine whether mitochondrial content can influence the pathogenesis of porphyria, we measured the mitochondrial DNA (mtDNA) copy number in the peripheral blood cells of 34 patients and 37 healthy individuals. We found that all AIP patients had a low number of mitochondria, likely as a result of a protective mechanism against an inherited heme synthesis deficiency. Furthermore, we identified a close correlation between disease penetrance and decreases in the mitochondrial content and serum levels of PERM1, a marker of mitochondrial biogenesis. In a healthy individual, mitochondrial count is usually modulated to fit its ability to respond to various environmental stressors and bioenergetic demands. In AIP patients, coincidentally, the phenotype only manifests in response to endogenous and exogenous triggers factors. Therefore, these new findings suggest that a deficiency in mitochondrial proliferation could affect the individual responsiveness to stimuli, providing a new explanation for the variability in the clinical manifestations of porphyria. However, the metabolic and/or genetic factors responsible for this impairment remain to be identified. In conclusion, both mtDNA copy number per cell and mitochondrial biogenesis seem to play a role in either inhibiting or promoting disease expression. They could serve as two novel biomarkers for porphyria.