Clinical applications of volatilomic assays.

The study of metabolomics is revealing immense potential for diagnosis, therapy monitoring, and understanding of pathogenesis processes. Volatilomics is a subcategory of metabolomics interested in the detection of molecules that are small enough to be released in the gas phase. Volatile compounds produced by cellular processes are released into the blood and lymph, and can reach the external environment through different pathways, such as the blood-air interface in the lung that are detected in breath, or the blood-water interface in the kidney that leads to volatile compounds detected in urine. Besides breath and urine, additional sources of volatile compounds such as saliva, blood, feces, and skin are available. Volatilomics traces its roots back over fifty years to the pioneering investigations in the 1970s. Despite extensive research, the field remains in its infancy, hindered by a lack of standardization despite ample experimental evidence. The proliferation of analytical instrumentations, sample preparations and methods of volatilome sampling still make it difficult to compare results from different studies and to establish a common standard approach to volatilomics. This review aims to provide an overview of volatilomics' diagnostic potential, focusing on two key technical aspects: sampling and analysis. Sampling poses a challenge due to the susceptibility of human samples to contamination and confounding factors from various sources like the environment and lifestyle. The discussion then delves into targeted and untargeted approaches in volatilomics. Some case studies are presented to exemplify the results obtained so far. Finally, the review concludes with a discussion on the necessary steps to fully integrate volatilomics into clinical practice.

SARS-CoV-2 infection triggers more potent antibody-dependent cellular cytotoxicity (ADCC) responses than mRNA-, vector-, and inactivated virus-based COVID-19 vaccines.

Neutralizing antibodies (NAbs) are elicited after infection and vaccination and have been well studied. However, their antibody-dependent cellular cytotoxicity (ADCC) functionality is still poorly characterized. Here, we investigated ADCC activity in convalescent sera from infected patients with wild-type (WT) severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) or omicron variant compared with three coronavirus disease 2019 (COVID-19) vaccine platforms and postvaccination breakthrough infection (BTI). We analyzed ADCC activity targeting SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in convalescent sera following WT SARS-CoV-2-infection (n = 91), including symptomatic and asymptomatic infections, omicron-infection (n = 8), COVID-19 vaccination with messenger RNA- (mRNA)- (BNT162b2 or mRNA-1273, n = 77), adenovirus vector- (n = 41), and inactivated virus- (n = 46) based vaccines, as well as post-mRNA vaccination BTI caused by omicron (n = 28). Correlations between ADCC, binding, and NAb titers were reported. ADCC was elicited within the first month postinfection and -vaccination and remained detectable for ≥3 months. WT-infected symptomatic patients had higher S-specific ADCC levels than asymptomatic and vaccinated individuals. Also, no difference in N-specific ADCC activity was seen between symptomatic and asymptomatic patients, but the levels were higher than the inactivated vaccine. Notably, omicron infection showed reduced overall ADCC activity compared to WT SARS-CoV-2 infection. Although post-mRNA vaccination BTI elicited high levels of binding and NAbs, ADCC activity was significantly reduced. Also, there was no difference in ADCC levels across the four vaccines, although NAbs and binding antibody titers were significantly higher in mRNA-vaccinated individuals. All evaluated vaccine platforms are inferior in inducing ADCC compared to natural infection with WT SARS-CoV-2. The inactivated virus-based vaccine can induce N-specific ADCC activity, but its relevance to clinical outcomes requires further investigation. Our data suggest that ADCC could be used to estimate the extra-neutralization level against COVID-19 and provides evidence that vaccination should focus on other Fc-effector functions besides NAbs. Also, the decreased susceptibility of the omicron variant to ADCC offers valuable guidance for forthcoming efforts to identify the specific targets of antibodies facilitating ADCC.

Circulating calprotectin as a potential biomarker of persistent olfactory dysfunctions in Post-COVID-19 patients.

BACKGROUND: This longitudinal prospective study aims to investigate the potential of circulating calprotectin (cCLP) as a biomarker in persistent olfactory dysfunctions following COVID-19 infection. METHODS: Thirty-six patients with persistent hyposmia or anosmia post COVID-19 were enrolled (HT0) and re-evaluated after three months of olfactory training (HT1). Two control groups included 18 subjects without olfactory defects post COVID-19 (CG1) and 18 healthy individuals (CG2). Nasal brushing of the olfactory cleft and blood collection were performed to assess circulating calprotectin levels. RESULTS: Higher calprotectin levels were observed in serum and nasal supernatant of hyposmic patients (HT0) compared to control groups (CG1 and CG2). Post-olfactory training (HT1), olfactory function improved significantly, paralleled by decreased calprotectin levels in serum and nasal samples. Circulating calprotectin holds potential as a biomarker in persistent olfactory dysfunctions after COVID-19. The decrease in calprotectin levels post-olfactory training implies a role in monitoring and evaluating treatment responses. DISCUSSION AND CONCLUSIONS: These findings contribute to the growing literature on potential biomarkers in post-COVID-19 olfactory dysfunctions and underscore the importance of investigating novel biomarkers for personalized patient management. Nevertheless, further studies are needed to validate the application of calprotectin assay in nasal diseases and its correlation with nasal cytology.

HTA model for laboratory medicine technologies: overview of approaches adopted in some international agencies.

The Health Technology Assessment (HTA) Working Group of the Emerging Technology Division of International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) aims to develop a methodological approach for producing structured HTA information for laboratory medicine technologies. This approach seeks to support decision-making processes at the country, regional, and/or hospital levels regarding the introduction of specific technologies. The focus of this model will primarily be on defining assessment elements within the domains of 'organizational aspects' and 'costs and economic evaluations', potentially differentiated by the type of diagnostic technology (e.g., genetic tests, molecular tests). To achieve this project's goal, a literature review and examination of websites of international HTA agencies have been conducted. The research aims to identify multidisciplinary methodological approaches used to assess laboratory diagnostic technologies and to pinpoint the domains and assessment elements utilized. We found 7 methodological articles describing methodological approaches adopted to assess laboratory diagnostic technologies. Among the HTA organizations considered, 23 reports were found, of which 7 were produced by the European Network of HTA (EUnetHTA), 4 by the National Institute for Health and Care Excellence Diagnostic Assessment Program (NICE DAP), and 12 by other HTA agencies. The EUnetHTA reports were rapid collaborative assessments covering various domains, while the NICE DAP reports focused on diagnostic guidances, including descriptions of technologies, clinical need and practice, diagnostic tests, accuracy, effectiveness, and cost-effectiveness. Finally, a survey targeting laboratory professionals will be conducted to introduce assessment elements, differentiated by the type of diagnostic technology, primarily for organizational and economic domains.

Laboratory Diagnosis of Intrathecal Synthesis of Immunoglobulins: A Review about the Contribution of OCBs and K-index.

The diagnosis of MS relies on a combination of imaging, clinical examinations, and biological analyses, including blood and cerebrospinal fluid (CSF) assessments. G-Oligoclonal bands (OCBs) are considered a "gold standard" for MS diagnosis due to their high sensitivity and specificity. Recent advancements have involved the introduced of kappa free light chain (k-FLC) assay into cerebrospinal fluid (CSF) and serum (S), along with the albumin quotient, leading to the development of a novel biomarker known as the "K-index" or "k-FLC index". The use of the K-index has been recommended to decrease costs, increase laboratory efficiency, and to skip potential subjective operator-dependent risk that could happen during the identification of OCBs profiles. This review aims to provide a comprehensive overview and analysis of recent scientific articles, focusing on updated methods for MS diagnosis with an emphasis on the utility of the K-index. Numerous studies indicate that the K-index demonstrates high sensitivity and specificity, often comparable to or surpassing the diagnostic accuracy of OCBs evaluation. The integration of the measure of the K-index with OCBs assessment emerges as a more precise method for MS diagnosis. This combined approach not only enhances diagnostic accuracy, but also offers a more efficient and cost-effective alternative.

Potential Anti-Inflammatory and Anti-Fatigue Effects of an Oral Food Supplement in Long COVID Patients.

Long coronavirus disease (COVID) syndrome leads to chronic inflammatory state onset that can have a multisystem impact and compromise organ function. Moreover, long COVID syndrome is often characterized by the presence of chronic fatigue, which affects subjects' daily activities and worsens their quality of life. The aim of our double-blind, placebo-controlled randomized trial (protocol code RS 150.21, approved on 4 November 2021) was to evaluate the beneficial effects of the consumption of 2 cps/day, for two months, of an oral food supplement (OFS), based on Echinacea angustifolia, rosehip, propolis, royal jelly and zinc, in long COVID patients, compared to a two-month placebo period. The OFS's vitamin C content was equal to 22.17 mg/g (8.87 mg/capsule). The OFS's total polyphenol content was 43.98 mg/g gallic acid equivalents. At the end of the in vivo study, we highlighted a significant decrease in the inflammatory parameters in the OFS period, compared to the placebo period (neutrophil-to-lymphocyte ratio, p = 0.0455; monocyte to-lymphocyte ratio, p = 0.0005; C-reactive protein, p = 0.0145). Our study also highlighted a significant increase in vitamin D serum values (p = 0.0005) and, at the same time, an improvement in patients' life quality and a reduction in fatigue, monitored by the fatigue severity scale. This study showed the OFS's beneficial effects on the inflammatory state, fatigue and quality of life in long COVID patients.

Vitamin D Deficiency in Professional Football Players during Competitive Season of Italian First Division (Serie A).

BACKGROUND: Data in the literature have demonstrated the crucial role that vitamin D plays in the human organism, and recent studies also emphasize this essential role of vitamin D in athletes. Indeed, vitamin D acts on the skeletal muscles and plays a fundamental role in numerous physiological processes involved in immune function. Many factors such as sun exposure, skin tone, body mass index and chronic illness affect vitamin D levels. The aim of the study is to evaluate vitamin D levels in professional football players in Italy and investigate the variations in vitamin D values in footballers who train at different latitudes. METHODS: The study performed is a retrospective observational study analyzing 25-OH vitamin D values in professional football players of the Italian First Division (Serie A). Two teams during the competitive season were selected: team A (latitude of 41° N in southern Italy) and team B (latitude of 45° N in northern Italy). Three time periods were identified and were classified as follows: the first quarter (May, June, July, and August), the second quarter (September, October, November, and December) and the third quarter (January, February, March, and April). The purpose of this was to study the average values of vitamin D during the year corresponding to different levels of sunlight exposure. Each athlete was subjected to at least one sampling during the three quarters of the competitive season. RESULTS: Both vitamin D insufficiency (10.1%) and overt deficiency (1.93%) were found in Italian Serie A players. Insufficient vitamin D values are between 20 ng/mL and 29 ng/mL and overt deficiency values <20 ng/mL. At the same time, the data demonstrated a significant variation in vitamin D values depending on the period of the competitive season and the latitude of the cities of the two teams. In detail, there was no significant difference in the first quarter, while there was a significant increase in vitamin D values in team B in the second and third quarter, at p < 0.01 and p < 0.05, respectively. CONCLUSIONS: Latitude and seasons have a significant impact on vitamin D levels. Therefore, it is essential to measure vitamin D in professional football players, especially during the spring and winter months, so as to monitor changes in levels in relation to the season and latitude and evaluate any supplements. Further studies should be performed to evaluate the relationship between vitamin D deficiency and football players' athletic performance.

Alteration of circadian sleep-wake rhythm and salivary melatonin secretion in idiopathic/isolated REM sleep behavior disorder: Preliminary evidence.

OBJECTIVE/BACKGROUND: Idiopathic/isolated REM sleep behavior disorder (iRBD) is widely regarded as an early sign of neurodegeneration leading to synucleinopathies. While circadian rhythm alterations in iRBD have been preliminarily demonstrated, evidence on melatonin secretion patterns in this clinical condition is limited. To address this knowledge gap, this exploratory study aimed to integrate salivary melatonin measurement with actigraphic monitoring in individuals with iRBD and age-matched healthy controls (HC) under real-life conditions. METHODS: Participants diagnosed with iRBD and HC underwent clinical evaluation and wore an actigraph for seven days and nights. Salivary melatonin concentrations were measured at five time points during the last night of recording. Comparative analyses were conducted on clinical data, actigraphic parameters, and melatonin levels between the two groups. RESULTS: iRBD participants (n = 18) showed greater motor (p < 0.01) and non-motor symptoms (p < 0.001), alongside disruptions in circadian sleep-wake rhythm compared to HC (n = 10). Specifically, actigraphy revealed a delayed central phase measurement (p < 0.05), reduced activity during the most active hours (p < 0.001), and decreased relative amplitude (p < 0.05). Total salivary melatonin concentration was significantly lower in iRBD (p < 0.05), with a slight but non-significant phase delay in dim light melatonin onset. CONCLUSIONS: This exploratory study highlights a dysregulation of circadian sleep-wake rhythm coupled with reduced melatonin secretion in iRBD. Future research could add to these preliminary findings to evaluate novel treatment approaches to regulate the sleep-wake cycle and elucidate the implications of circadian dysregulation in the conversion from iRBD to neurodegeneration.

The Effects of Ultrasonic Scaling and Air-Abrasive Powders on the Topography of Implant Surfaces: Scanning Electron Analysis and In Vitro Study.

OBJECTIVES: This in vitro study aimed to investigate the impact of bicarbonate air-abrasive powders and ultrasonic scaling with stainless steel tips on the micro- and nanotopography and roughness of three different implant-abutment junction titanium surfaces. MATERIALS AND METHODS: Three types of sterile and decontaminated titanium surfaces (RS, UTM, XA) were used for analysis. Nine disks per surface type were subjected to micro- and nanotopography analysis, scanning electron microscopy (SEM), roughness analysis, and fibroblast cultivation. Ultrasonic debridement and air polishing were performed on the surfaces. Human dermal fibroblasts were cultured on the surfaces for 5 days. STATISTICAL ANALYSIS: Data analysis adhered to ISO 25178 standards for surface texture assessment. SEM micrographs were used to reconstruct areas for extracting roughness parameters. Excel and Mex 6.0 software were utilized for quantitative and stereoscopic analysis. RESULTS: The study found varying effects on surface roughness posttreatment. RS Disco samples exhibited higher surface roughness compared with UTM and XA samples, both in average and nanoscale roughness. Decontamination led to increased surface roughness for all samples, particularly RS Disco. Fibroblast growth tests revealed enhanced cell network formation on decontaminated discs, possibly due to increased nanoscale roughness or the presence of bicarbonate salts. CONCLUSION: The study underscores the complex interplay between surface topography, microbial biofilm, and treatment efficacy in peri-implant disease management. While smoother surfaces may resist biofilm accumulation, increased nanoscale roughness postdecontamination can enhance fibroblast attachment and soft tissue integration. This dichotomy highlights the need for tailored treatment protocols that consider material-specific factors, emphasizing that successful implant therapy should balance microbial control with conducive surface characteristics for long-term osseointegration and soft tissue stability.

Autoantibody profiles assessment in individuals with persistent olfactory impairment following SARS-CoV-2 infection.

BACKGROUND: Olfactory impairment, particularly hyposmia and anosmia, has emerged as a distinctive early symptom of SARS-CoV-2. Drawing on the historical association of autoimmune diseases with olfactory function, this study delves into the connections between COVID-19, autoimmunity, and persistent olfactory dysfunctions, focusing on individuals experiencing long-lasting smell disorders (3-18 months post-SARS-CoV-2 infection). METHODS: The study comprised 36 Long Covid patients with persistent olfactory dysfunctions, alongside two control groups. Olfactory functionality was assessed using the Sniffin' Sticks extended test. Non-invasive olfactory mucosa brushing and nasal secretions were processed for nasal samples, while serum samples were obtained through peripheral venous sampling. A panel of autoantibodies, including Immunocirculating Complexes, ANA, ENA, and AECA, was investigated in serum and brush supernatant samples. RESULTS: Contrary to expectations, the absence of traditional autoantibodies challenges the proposed autoimmune etiology of Long Covid-associated olfactory dysfunction. However, the presence and potential pathogenic role of AECA suggest viral cytopathic and inflammatory involvement in specific anatomical districts. One hypothesis explores the impact of inflammation and cytokine release induced by the viral infection, altering neuronal signaling and contributing to persistent hyposmia. CONCLUSION: This research contributes to our understanding of the complex relationships between autoimmunity, olfactory impairment, and COVID-19. The absence of classical autoantibodies challenges prevailing theories, while the prominence of AECA hints at unique viral-induced pathogenic mechanisms. By unraveling these complexities, this study enhances our comprehension of post-acute sequelae, offering valuable perspectives on immune-mediated responses in the aftermath of the pandemic.

Characterization and Clinical Assessment of a Peculiar Case of Hemolytic Anemia.

Thalassemic diseases are characterized by a reduced (ß+) or absent (ß0) synthesis of the globin chains of hemoglobin (Hb) due to genetic mutations. ß-thalassemia was more frequent in the Mediterranean area, but now it is diffused worldwide. Three possible genetic forms can be distinguished: ß0/ß0, the most severe (Cooley's disease); ß0/ß+ of intermediate severity; ß+/ß+ associated with ß-thalassemia intermedia or minor. Recently, a clinical non-genetic classification has been proposed: transfusion-dependent thalassemia (TDT), requiring regular lifetime blood transfusions, and non-transfusion-dependent thalassemia (NTDT), requiring occasional transfusions to manage acute cases. In this report, we studied a patient whose blood count indicated a severe anemia but also showed thrombocytosis, leukocytosis, and an elevated number of nucleated red blood cells (NRBC). These altered blood parameters suggested initially a possible diagnosis of hemoglobinopathy or myeloproliferative syndrome. The molecular and genetic analyses demonstrated the presence of HbF (5.3%) and HbA2 (7.7%) and the presence of the homozygote mutation (IVS1.6T>C) in the ß-globin gene. According to these data, a diagnosis of ß-thalassemia intermedia form has been proposed. Nevertheless, the clinical condition, the presence of thrombocytosis, leukocytosis, an elevated number of NRBC, and the frequent blood transfusions lead to reclassification of the patient as TDT subject. Consequently, this result suggests that a unique genotype-phenotype correlation is not possible in the presence of ß+mutations since other concomitant pathologies can exacerbate the disease.

CSF dynamics of orexin and ß-amyloid42 levels in narcolepsy and Alzheimer's disease patients: a controlled study.

ß-amyloid42 (Aß42) in Alzheimer's disease (AD) and orexin in narcolepsy are considered crucial biomarkers for diagnosis and therapeutic targets. Recently, orexin and Aß cerebral dynamics have been studied in both pathologies, but how they interact with each other remains further to be known. In this study, we investigated the reliability of using the correlation between orexin-A and Aß42 CSF levels as a candidate marker to explain the chain of events leading to narcolepsy or AD pathology. In order to test the correlation between these biomarkers, patients diagnosed with AD (n = 76), narcolepsy type 1 (NT1, n = 17), narcolepsy type 2 (NT2, n = 23) and healthy subjects (n = 91) were examined. Patients and healthy subjects underwent lumbar puncture between 8:00 and 10:00 am at the Neurology Unit of the University Hospital of Rome "Tor Vergata". CSF levels of Aß42, total-tau, phosphorylated-tau, and orexin-A were assessed. The results showed that CSF levels of Aß42 were significantly lower (p < 0.001) in AD (332.28 ± 237.36 pg/mL) compared to NT1 (569.88 ± 187.00 pg/mL), NT2 (691.00 ± 292.63 pg/mL) and healthy subjects (943.68 ± 198.12 pg/mL). CSF orexin-A levels were statistically different (p < 0.001) between AD (148.01 ± 29.49 pg/mL), NT1 (45.94 ± 13.63 pg/mL), NT2 (104.92 ± 25.55 pg/mL) and healthy subjects (145.18 ± 27.01 pg/mL). Moderate-severe AD patients (mini mental state examination < 21) showed the highest CSF orexin-A levels, whereas NT1 patients showed the lowest CSF orexin-A levels. Correlation between CSF levels of Aß42 and orexin-A was found only in healthy subjects (r = 0.26; p = 0.01), and not in narcolepsy or AD patients. This lack of correlation in both diseases may be explained by the pathology itself since the correlation between these two biomarkers is evident only in the healthy subjects. This study adds to the present literature by further documenting the interplay between orexinergic neurotransmission and cerebral Aß dynamics, possibly sustained by sleep.

Comparable antibody levels in heterologous and homologous mRNA COVID-19 vaccination, with superior neutralizing and IgA antibody responses in mRNA homologous boosting.

BACKGROUND: Priming with two doses of AZD1222 (Oxford-AstraZeneca; ChAd) followed by a third mRNA vaccine boosting is considered in several countries, yet comparisons between heterologous and homologous booster efficacy remain unexplored. AIM: To evaluate and contrast the immunogenicity of homologous and heterologous boosting regimens. METHOD: The study examined antibody responses in 1113 subjects, comprising 895 vaccine-naïve individuals across different vaccination strategies (partial, primary series, heterologous booster, homologous booster) and 218 unvaccinated, naturally infected individuals. Assessments included neutralizing total antibodies (NTAbs), total antibodies (TAbs), anti-S-RBD IgG, and anti-S1 IgA levels. RESULTS: The study found mRNA vaccines to exhibit superior immunogenicity in primary series vaccination compared to ChAd, with mRNA-1273 significantly enhancing NTAbs, TAbs, anti-S-RBD IgG, and anti-S1 IgA levels (p < 0.001). Both booster types improved antibody levels beyond primary outcomes, with no significant difference in TAbs and anti-S-RBD IgG levels between regimens. However, homologous mRNA boosters significantly outperformed heterologous boosters in enhancing NTAbs and anti-S1 IgA levels, with the BNT/BNT/BNT regimen yielding particularly higher enhancements (p < 0.05). CONCLUSION: The study concludes that although TAbs and anti-S-RBD IgG antibody levels are similar for both regimens, homologous mRNA boosting outperform heterologous regimen by enhancing anti-S1 IgA and neutralizing antibody levels.

Follow-Up and Comparative Assessment of SARS-CoV-2 IgA, IgG, Neutralizing, and Total Antibody Responses After BNT162b2 or mRNA-1273 Heterologous Booster Vaccination.

BACKGROUND: Priming with ChAdOx1 followed by heterologous boosting is considered in several countries. Nevertheless, analyses comparing the immunogenicity of heterologous booster to homologous primary vaccination regimens and natural infection are lacking. In this study, we aimed to conduct a comparative assessment of the immunogenicity between homologous primary vaccination regimens and heterologous prime-boost vaccination using BNT162b2 or mRNA-1273. METHODS: We matched vaccinated naïve (VN) individuals (n = 673) with partial vaccination (n = 64), primary vaccination (n = 590), and primary series plus mRNA vaccine heterologous booster (n = 19) with unvaccinated naturally infected (NI) individuals with a documented primary SARS-CoV-2 infection (n = 206). We measured the levels of neutralizing total antibodies (NTAbs), total antibodies (TAbs), anti-S-RBD IgG, and anti-S1 IgA titers. RESULTS: Homologous primary vaccination with ChAdOx1 not only showed less potent NTAb, TAb, anti-S-RBD IgG, and anti-S1 IgA immune responses compared to primary BNT162b2 or mRNA-1273 vaccination regimens (p < 0.05) but also showed ~3-fold less anti-S1 IgA response compared to infection-induced immunity (p < 0.001). Nevertheless, a heterologous booster led to an increase of ~12 times in the immune response when compared to two consecutive homologous ChAdOx1 immunizations. Furthermore, correlation analyses revealed that both anti-S-RBD IgG and anti-S1 IgA significantly contributed to virus neutralization among NI individuals, particularly in symptomatic and pauci-symptomatic individuals, whereas among VN individuals, anti-S-RBD IgG was the main contributor to virus neutralization. CONCLUSION: The results emphasize the potential benefit of using heterologous mRNA boosters to increase antibody levels and neutralizing capacity particularly in patients who received primary vaccination with ChAdOx1.