Analytical and clinical evaluations of SNIBE Maglumi chemiluminescent immunoassay for the detection of SARS-CoV-2 antigen in salivary samples.

OBJECTIVES: In this study, we describe the analytical and clinical performances of the SNIBE Maglumi SARS-CoV-2 antigen fully-automated chemiluminescent immunoassay (MAG-CLIA) on salivary samples. METHODS: Limit of detection (LOD), linearity and precision were tested for values close to or below the declared LOD. Clinical performance of MAG-CLIA was evaluated on leftover salivary samples from the healthcare workers (HCW) surveillance program, at the University-Hospital of Padova. Salivary samples were analyzed by Lumipulse G SARS-CoV-2 Ag, and in case where the values exceeded 0.41 ng/L, further testing was conducted using TaqPathTM COVID-19 RT-PCR (Applied Biosystems, Thermo Fisher Scientific). RESULTS: The estimated MAG-CLIA LOD was 3 ng/L, with repeatability of 7.5 %. Good linearity was demonstrated by diluting two samples at 52.7 ng/L and 211.4 ng/L. Of the 228 HCW samples, 59/228 (25.9 %) were positive, 169/228 (74.1 %) were negative. MAG-CLIA SARS-CoV-2 sAg median level (and interquartile range [IQR]) was 5.03 ng/L (<0.001-35.8 ng/L) for positive and <0.001 ng/L (<0.001 ng/L) for negative samples. MAG-CLIA AUC was 0.795 (95 % CI: 0.720-0.871). Using the best cut-off, 3.5 ng/L, sensitivity and specificity were 57.1 % (95 % CI: 42.2-71.2 %) and 97.0 % (95 % CI: 93.2-99.0 %), respectively. The agreement with the molecular assay was 88.1 % (Cohen's kappa 0.606 [SE=0.066, p<0.001]). CONCLUSIONS: The analytical performances of MAG-CLIA are satisfactory, also when values below LOD were tested. In saliva samples, although specificity was elevated, clinical performance was not comparable with that on nasopharyngeal swabs (NPS).

New insights into SARS-CoV-2 Lumipulse G salivary antigen testing: accuracy, safety and short TAT enhance surveillance.

OBJECTIVES: The rapid, accurate and safe detection of SARS-CoV-2 is the key to improving surveillance and infection containment. The aim of the present study was to ascertain whether, after heat/chemical inactivation, SARS-CoV-2 N antigen chemiluminescence (CLEIA) assay in saliva remains a valid alternative to molecular testing. METHODS: In 2022, 139 COVID-19 inpatients and 467 healthcare workers were enrolled. In 606 self-collected saliva samples (Salivette), SARS-CoV-2 was detected by molecular (TaqPath rRT-PCR) and chemiluminescent Ag assays (Lumipulse G). The effect of sample pre-treatment (extraction solution-ES or heating) on antigen recovery was verified. RESULTS: Salivary SARS-CoV-2 antigen assay was highly accurate (AUC=0.959, 95% CI: 0.943-0.974), with 90% sensitivity and 92% specificity. Of the 254 antigen positive samples, 29 were false positives. We demonstrated that heterophilic antibodies could be a cause of false positive results. A significant antigen concentration decrease was observed after ES treatment (p=0.0026), with misclassification of 43 samples. Heat had a minimal impact, after treatment the correct classification of cases was maintained. CONCLUSIONS: CLEIA SARS-CoV-2 salivary antigen provides accurate, timely and high-throughput results that remain accurate also after heat inactivation, thus ensuring a safer work environment. This supports the use of salivary antigen detection by CLEIA in surveillance programs.

The University of Padua salivary-based SARS-CoV-2 surveillance program minimized viral transmission during the second and third pandemic wave.

BACKGROUND: The active surveillance of students is proposed as an effective strategy to contain SARS-CoV-2 spread and prevent schools' closure. Saliva for molecular testing is as sensitive as naso-pharyngeal swab (NPS), self-collected and well accepted by participants. This prospective study aimed to verify whether the active surveillance of the Padua University employees by molecular testing of self-collected saliva is an effective and affordable strategy for limiting SARS-CoV-2 spread. METHODS: A surveillance program based on self-collection of saliva every 2 weeks (October 2020-June 2021) was conducted. Among 8183 employees of the Padua University, a total of 6284 subjects voluntarily took part in the program. Eight collection points guaranteed the daily distribution and collection of barcoded salivary collection devices, which were delivered to the laboratory by a transport service for molecular testing. Quarantine of positive cases and contact tracing were promptly activated. RESULTS: Among 6284 subjects, 206 individuals were SARS-CoV-2 positive (99 by salivary testing; 107 by NPS performed for contact tracing or symptoms). The cumulative SARS-CoV-2 incidence in this cohort was 3.1%, significantly lower than that of employees not in surveillance (8.0%), in Padua (7.1%) and in the Veneto region (7.2%). Employees with positive saliva results were asymptomatic or had mild symptoms. The levels of serum antibodies after 3 months from the infection were correlated with age and Ct values, being higher in older subjects with greater viral loads. CONCLUSIONS: Salivary-based surveillance with contact tracing effectively allowed to limit SARS-CoV-2 contagion, also in a population with a high incidence.

Hyris bCUBE SARS-CoV-2 rapid molecular saliva testing: a POCT innovation on its way.

OBJECTIVES: The reliable identification of individuals with SARS-CoV-2 infection is the cornerstone for containing viral spread. Rapid molecular point-of-care testing (POCT) of saliva might reduce analysis time, thus increasing the efficacy of contact tracing. In this study, a new POCT RT-PCR assay for the detection of SARS-CoV-2 RNA in saliva was evaluated and compared with an already validated CE-IVD method. METHODS: An evaluation was made of 160 left-over salivary samples (27 frozen, kept at -80 °C and 133 fresh), collected using Salivette (Sarstedt, Germany). Samples were analyzed by TaqPath COVID-19 CE-IVD RT-PCR kit, QuantStudio5 Real-Time (Applied Biosystems, USA) (TaqPath) and bKIT Virus Finder COVID-19 Saliva (Hyris Global Diagnostics, Italy). Performances of three- and fivefold pooling strategies were also evaluated. Blood assay interference in saliva was also tested with Hyris. RESULTS: On using TaqPath, SARS-CoV-2 positivity was detected in 35 samples. Another 10 positive samples were artificially-generated by blind mixing of positive with negative samples. Hyris positive and negative percentages of agreement were 97.6 (95% CI: 87.2-99.9%) and 100 (95% CI: 97.0-100%), respectively. Seventeen positive pools, evaluated for threefold strategy, were all correctly determined by both systems. For the 5-pool strategy, 94.7% (18/19) of samples resulted positive with the Hyris system, and 100% with TaqPath. The presence of 1% of blood (v/v) in saliva did not interfere with the accuracy of Hyris assay. CONCLUSIONS: The sensitivity and specificity of the bKIT Virus Finder COVID-19 Saliva were optimal with respect to TaqPath. In view of the safe and straightforward pre-analytical procedure involved, and the small size of the Hyris bCube, the Hyris system can be used for POCT.

SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics.

Objectives: The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results. Methods: Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other. Results: A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%). Conclusions: This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Lipopolysaccharide-induced chorioamnionitis and postnatal lung injury: The beneficial effects of L-citrulline in newborn rats.

AIM OF THE STUDY: The lung architecture of newborns appears to be affected by an inflammatory reaction to maternal choriodecidual layer infection. L-citrulline (L-Cit) was administered to pregnant rats exposed to intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis to investigate its effect on neonatal lung injury. MATERIALS AND METHODS: The pups were assigned to four experimental groups: 1- pups exposed to intra-amniotic NaCl but not to postnatal L-Cit (Controls); 2 - pups exposed to intra-amniotic NaCl as well as to postnatal L-Cit treatment (L-Cit group); 3 - pups exposed to prenatal LPS but not to postnatal (LPS); 4- pups exposed to prenatal LPS as well as to postnatal L-Cit treatment (LPS + L-Cit). Some pups in each group were sacrificed on postnatal (P) day 3 and others on day 7. The pups' lungs were harvested for morphometric analysis; cytokine, arginase 1, and VEGF values were quantified. Serum arginine, citrulline, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine, NG-monomethyl arginine, and homoarginine levels were determined using UPLC-MS/MS. RESULTS: L-Cit attenuated the disruption of alveolar growth in the LPS + L-Cit group. Arginine, homo-arginine, and ADMA levels fell in the LPS treated groups. Arginine and ADMA rose at P7 in the L-Cit group whose members also showed higher VEGF levels with respect to the Controls. The Controls, instead, showed higher IL-10 and IL-1ß values with respect to the L-Cit group at P7. Arginase 1 was higher in the LPS groups with respect to the Controls at P7. CONCLUSIONS: L-Cit improved alveolar and vascular growth diminishing the lung inflammatory response in the newborn rats exposed to intra-amniotic LPS. The ADMA/DDAH/NO pathway appeared to counteract proinflammatory cytokine production and to sustain macrophage migration.

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFß1.

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFß1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. RESULTS: NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFß1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFß1 canonical signaling pathway. TGFß1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFß1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFß1 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFß1 on cell signaling, and the formation of complexes between TGFß1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

Defective ADAMTS13 synthesis as a possible consequence of NASH in an obese patient with recurrent thrombotic thrombocytopenic purpura.

OBJECTIVES: Thrombotic thrombocytopenic purpura (TTP) is a rare and devastating hematologic disorder frequently associated with multiple organ failure and sometimes death. This syndrome is mainly associated with severe deficiency of ADAMTS13, a disintegrin and metalloprotease with thrombospondin (TSP)-1 repeats, cleaving high molecular weight von Willebrand Factor (ULVWF) multimers. Decreased plasma ADAMTS13 activity results in the accumulation of ULVWF multimers with consequent platelet activation. Recently, obesity has been considered as a potential independent risk factor for TTP, but the reason of this association is still unknown. METHODS AND RESULTS: We describe an unusual case of fatal recurrent TTP in a morbid obese female with non-alcoholic steatohepatitis (NASH) and severe ADAMTS13 activity deficiency due neither to an inhibitory autoantibody nor to a gene mutation. CONCLUSIONS: Visceral obesity is associated with non-alcoholic fatty liver disease (NAFLD) and NASH: we hypothesized that these conditions can influence ADAMTS13 antigen and activity. In fact, hepatic stellate cells (HSC) are the main producers of ADAMTS13, and a decrease in ADAMTS13 activity has been reported in liver disease.

Evaluation of a new molecular test for the detection of SARS-CoV-2 nucleic acid in salivary samples.

Background: Molecular testing is considered the gold standard for the detection of SARS-CoV-2. This study aimed to compare the performance of the P742H SARS-CoV-2 Nucleic Acid Multiplex Detection Kit in salivary samples, with respect to the 732HF Novel Coronavirus (2019-nCoV) Nucleic Acid Detection Kit and the TaqPath COVID-19 CEIVD RT-PCR Kit, used at University-Hospital of Padova, Italy. Methods: One hundred twenty-four salivary samples selfcollected by healthcare workers (HCW) during the screening program at University-Hospital of Padova, Italy, from Oct to Nov 2022, were included in the study. RNA extraction was performed by Viral DNA and RNA Extraction Kit (Technogenetics, Lodi, Italy) and amplification by P742H and 732HF (Technogenetics, Lodi, Italy). RNA was extracted using MagNa Pure 96 DNA and Viral NA Small Volume Kit (Roche, Switzerland) for TaqPath analysis (Thermo Fisher Scientific, USA).

Anti-VEGF therapy selects for clones resistant to glucose starvation in ovarian cancer xenografts.

BACKGROUND: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis. METHODS: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy. RESULTS: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation. CONCLUSION: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy.

Effectiveness of the combined evaluation of KLK3 genetics and free-to-total prostate specific antigen ratio for prostate cancer diagnosis.

PURPOSE: Of serum prostate specific antigen variability 40% depends on inherited factors. We ascertained whether the knowledge of KLK3 genetics would enhance prostate specific antigen diagnostic performance in patients with clinical suspicion of prostate cancer. MATERIALS AND METHODS: We studied 1,058 men who consecutively underwent prostate biopsy for clinical suspicion of prostate cancer. At histology prostate cancer was present in 401 cases and absent in 657. Serum total prostate specific antigen and the free-to-total prostate specific antigen ratio were determined. Four polymorphisms of the KLK3 gene (rs2569733, rs2739448, rs925013 and rs2735839) and 1 polymorphism of the SRD5A2 gene (rs523349) were studied. The influence of genetics on prostate specific antigen variability was evaluated by multivariate linear regression analysis. The performance of total prostate specific antigen and the free-to-total prostate specific antigen ratio alone or combined with a genetically based patient classification were defined by ROC curve analyses. RESULTS: For prostate cancer diagnosis the free-to-total prostate specific antigen ratio index alone (cutoff 11%) was superior to total prostate specific antigen (cutoff 4 ng/ml) and to free-to-total prostate specific antigen ratio reflex testing (positive predictive value 61%, 43% and 54%, respectively). Prostate specific antigen correlated with KLK3 genetics (rs2735839 polymorphism p = 0.001, and rs2569733, rs2739448 and rs925013 haplotype combination p = 0.003). In patients with different KLK3 genetics 2 optimal free-to-total prostate specific antigen ratio cutoffs (11% and 14.5%) were found. For free-to-total prostate specific antigen ratio values between 11% and 14.5% the prostate cancer probability ranged from 30.0% to 47.4% according to patient genetics. CONCLUSIONS: The free-to-total prostate specific antigen ratio is superior to total prostate specific antigen for prostate cancer diagnosis, independent of total prostate specific antigen results. Free-to-total prostate specific antigen ratio findings below 11% are positively associated with prostate cancer and those above 14.5% are negatively associated with prostate cancer, while the interpretation of those between 11% and 14.5% is improved by patient KLK3 genetic analysis.

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N-terminal peptide (NT-S100A8).

After isolating NT-S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca(2+)](i) oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT-S100A8. In NT-S100A8 stimulated ß-TC6 (insulinoma cell line) culture medium, insulin and [Ca(2+)] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca(2+)](i) oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT-S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT-S100A8 induced a rapid insulin release and enhanced ß-TC6 [Ca(2+)](i) oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT-S100A8, [Ca(2+)] in ß-TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT-S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB-α, but it independently activated Akt and NF-κB signaling in PC cells. In conclusion, NT-S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF-κB signaling, NT-S100A8 enhances [Ca(2+)](i) oscillations and insulin release, probably by inducing Ca(2+) influx from the extracellular space, but it does not interfere with insulin signaling.

Mass spectrometry measurement of plasma hepcidin for the prediction of iron overload.

BACKGROUND: Hepcidin has emerged as the primary regulator of iron homeostasis. Previous studies on assessing urinary hepcidin are limited. We developed a method for quantifying hepcidin-25 (Hep-25) in plasma using surface-enhanced laser-desorption-ionization time-of-flight mass spectrometry (SELDI-TOF/MS) and a 25-AA peptide as reference standard. The aims of the study were 1) to assess the performance of this method in different conditions of iron metabolism disorders; 2) to assess the diagnostic validity of non-invasive serum biomarkers in the identification of iron overload. METHODS: Validation of the method was performed in 10 patients with type I hemochromatosis (HE) and in 177 subjects previously enrolled in a general population epidemiological study. Among the latter group, 17 had non-alcoholic fatty liver disease, 10 had chronic hepatitis C, and 150 subjects had normal ultrasound, normal liver function tests (LFTs), an alcohol intake < 20 g ethanol/day and were negative for the C282Y mutation. The following biomarkers were assayed in each case: plasma Hep-25, C282Y and H63D mutations of the HFE gene; serum iron, ferritin (SF), transferrin saturation, transaminases, γ-glutamyltransferase (GGT), glucose, insulin, total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglycerides. RESULTS: Plasma Hep-25 concentrations were higher in HCV+ patients (26.3 ± 7.2 nmol/L) than in controls, and correlated positively with SF (p < 0.001). H63D heterozygous subjects revealed a pattern of iron overload that was significantly higher than H63D wild type subjects. Analyzing the data with the Biomarker Pattern 5.0.2. software to identify the most significant biomarkers for discriminating between HE cases and controls allowed us to produce an algorithm with four terminal nodes, which included glucose > 4.8 mmol/L and Hep-25/SF ratio ≤ 6.6 as the main splitters. These variables enabled the correct diagnosis of HE with 100% sensitivity, 93% specificity and an area under the receiver operating characteristic (ROC) curve of 0.993. CONCLUSIONS: Our plasma Hep-25 mass spectrometry method yields measurements that reflect pathological and genetic influences; simple non-invasive biomarkers (Hep-25/SF ratio and glucose) can predict the presence of HE.

Salivary SARS-CoV-2 antigen rapid detection: A prospective cohort study.

BACKGROUND AND AIM: SARS-CoV-2 quick testing is relevant for the containment of new pandemic waves. Antigen testing in self-collected saliva might be useful. We compared salivary and naso-pharyngeal swab (NPS) SARS-CoV-2 antigen detection by a rapid chemiluminescent assay (CLEIA) and two different point-of-care (POC) immunochromatographic assays, with results of molecular testing. METHODS: 234 patients were prospectively enrolled. Paired self-collected saliva (Salivette) and NPS were obtained to perform rRT-PCR, chemiluminescent (Lumipulse G) and POC (NPS: Fujirebio and Abbott; saliva: Fujirebio) for SARS-CoV-2 antigen detection. RESULTS: The overall agreement between NPS and saliva rRT-PCR was 78.7%, reaching 91.7% at the first week from symptoms. SARS-CoV-2 CLEIA antigen was highly accurate in distinguishing positive and negative NPS (ROC-AUC = 0.939, 95%CI:0.903-0.977), with 81.6% sensitivity and 93.8% specificity. This assay on saliva reached the optimal value within 7 days from symptoms onset (Sensitivity: 72%; Specificity: 97%). Saliva POC antigen was limited in sensitivity (13%), performing better in NPS (Sensitivity: 48% and 66%; Specificity: 100% and 99% for Espline and Abbott respectively), depending on viral loads. CONCLUSIONS: Self-collected saliva is a valid alternative to NPS for SARS-CoV-2 detection by molecular, but also by CLEIA antigen testing, which is therefore potentially useful for large scale screening.

Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts.

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways-comprising both oxidative phosphorylation and glycolysis-in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment.

SARS-CoV-2 identification and IgA antibodies in saliva: One sample two tests approach for diagnosis.

AIM: This study aims to verify whether standardized saliva collection is suitable for SARS-CoV-2 molecular detection and IgA measurement. METHODS: 43 COVID-19 inpatients and 326 screening subjects underwent naso-pharyngeal (NP)-swab and saliva collection (Salivette). Inpatients also underwent repeated blood collections to evaluate inflammation and organs involvement. In all patients and subjects, SARS-CoV-2 (gene E) rRT-PCR was undertaken in saliva and NP-swabs. Salivary IgA and serum IgA, IgG, IgM were measured on inpatients' samples. RESULTS: NP-swabs and saliva were both SARS-CoV-2 positive in 7 (16%) or both negative in 35 (82%) out of 43 patients successfully included in the study. NP-swabs and saliva results did not perfectly match in one patient (saliva positive, NP-swab negative). Positive molecular results were significantly associated with disease duration (p = 0.0049). 326/326 screening subjects were SARS-CoV-2 negative on both NP-swabs and saliva. Among the 27 saliva samples tested for IgA, 18 were IgA positive. Salivary IgA positivity was associated with pneumonia (p = 0.002) and CRP values (p = 0.0183), not with other clinical and molecular data, or with serum immunoglubulins. CONCLUSIONS: A standardized saliva collection can be adopted to detect SARS-CoV-2 infection in alternative to NP-swabs. Preliminary data on salivary IgA support the use of saliva also for patient monitoring.

Antibodies against synthetic deamidated gliadin peptides for celiac disease diagnosis and follow-up in children.

BACKGROUND: AGA IgA II and AGA IgG II have recently been suggested as reliable tools for celiac disease (CD) diagnosis. We compared their utility for diagnosis and monitoring CD in children with that of tTG IgA, an established CD marker. METHODS: We studied a cohort of 161 CD and 129 control children in whom CD was histologically confirmed or ruled out. We followed 37 children with CD on a gluten-free diet for 12-84 months. In fasting sera, we measured AGA IgA II, AGA IgG II, and tTG IgA using ELISAs. RESULTS: The best sensitivity (92.5%), specificity (97.6%), positive predictive value (98%), and negative predictive value (91.2%) were obtained using tTG IgA. AGA IgG II correctly identified 3 of 3 children with CD with total IgA deficiency who had negative AGA IgA II and tTG IgA results. In children <2 years old without total IgA deficiency, AGA IgG II and tTG IgA performed equally well (sensitivity 96.4% and specificity 100%). AGA IgA II, AGA IgG II, and tTG IgA concentrations diminished significantly (P < 0.0001) after 1 year of a gluten-free diet, reaching values below the cutoff in 87%, 70%, and 51% of cases, respectively. CONCLUSIONS: The best available index for diagnosing CD in children was tTG IgA. In infants <2 years old, AGA IgG II performed as well as tTG IgA in cases without total IgA deficiency and allowed detection of CD when total IgA was <0.06 g/L. Gluten-free diet monitoring can be achieved using any of the studied serum markers.

Is vitamin D deficiency a risk factor for osteonecrosis of the jaw in patients with cancer? A matched case-control study.

PURPOSE: A previous case-control histomorphometric study showed higher odds of osteomalacia in patients with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Vitamin D deficiency causes osteomalacia and may therefore be involved in the pathogenesis of BRONJ. The present case-control study aimed at testing such hypothesis. MATERIALS AND METHODS: BRONJ+ and BRONJ- patients treated with bisphosphonates were matched by sex (same) and age (within 5 years). Serum 25-hydroxy-vitamin D (25-OH-D), parathyroid hormone, bone alkaline phosphatase, total procollagen type 1 amino-terminal propeptide, carboxy-terminal collagen crosslinks, Dickkopf WNT signaling pathway inhibitor 1 and sclerostin were measured. RESULTS: The main outcome was vitamin D deficiency defined as 25-OH-D < 50 nmol/l. A total of 51 BRONJ+ and 73 BRONJ- patients were studied. The frequency (95% CI) of vitamin D deficiency was 59% (45%-72%) in BRONJ+ and 62% (48%-75%) in BRONJ- patients. This amounts to a difference of -3% (-22%-16%, p = 0.77) for BRONJ+ patients. Serum 25-hydroxy-vitamin D and parathyroid hormone were similar in BRONJ+ and BRONJ- patients. Among the bone metabolism markers, only sclerostin differed between the two groups, being higher in BRONJ+ patients. CONCLUSION: The present matched case-control study suggests that vitamin D deficiency is not a risk factor for BRONJ.