Consecutive Serum Samples Research Articles

Risk of celiac disease autoimmunity is modified by interactions between CD247 and environmental exposures

Season of birth, viral infections, HLA haplogenotypes and non-HLA variants are implicated in the development of celiac disease and celiac disease autoimmunity, suggesting a combined role of genes and environmental exposures. The aim of the study was to further decipher the biological pathways conveying the season of birth effect in celiac disease autoimmunity to gain novel insights into the early pathogenesis of celiac disease. Interactions between season of birth, genetics, and early-life environmental factors on the risk of celiac autoimmunity were investigated in the multicenter TEDDY birth cohort study. Altogether 6523 genetically predisposed children were enrolled to long-term follow-up with prospective sampling and data collection at six research centers in the USA, Germany, Sweden and Finland. Celiac disease autoimmunity was defined as positive tissue transglutaminase antibodies in two consecutive serum samples. There was a significant season of birth effect on the risk of celiac autoimmunity. The effect was dependent on polymorphisms in CD247 gene encoding for CD3ζ chain of TCR-CD3 complex. In particular, children with major alleles for SNP rs864537A > G, in CD247 (AA genotype) had an excess risk of celiac autoimmunity when born March–August as compared to other months. The interaction of CD247 with season of birth on autoimmunity risk was accompanied by interactions with febrile infections between the ages of 3–6 months. Considering the important role of TCR-CD3 complex in the adaptive immune response and our findings here, CD247 variants and their possible effect of subgroups in autoimmunity development could be of interest in the design of future gene-environment studies of celiac disease. ClinicalTrials.gov Identifier: NCT00279318.

Diagnosis of Menopause in a Captive Sumatran Orangutan (Pongo abelii).

Humans were long thought to be the only mammal to experience menopause, the permanent cessation of reproduction followed by a long post-reproductive lifespan. More recently, evidence has been found for the existence of menopause in other long-lived mammals, including chimpanzees and gorillas. However, orangutans, which have the longest interbirth interval of any primate, have rarely been studied in this period of their lives. In this paper, we describe clinical, ultrasound, endocrine, and histological evidence consistent with a natural menopause in a captive, previously fertile, Sumatran orangutan (Pongo abelii), aged approximately 50. Consecutive serum samples showed low levels of estradiol and high levels of follicle-stimulating hormone. Transvaginal ultrasound revealed an atrophic uterus with an antero-posterior diameter of 2.36 cm, an endometrial thickness of 2 mm, and inactive ovaries. Following this female's death from a subdural hematoma, histological examination of the ovaries showed a dense stroma with corpora albicantia, in comparison to the numerous primordial follicles seen in the ovaries of a stillborn infant female orangutan. These multiple lines of evidence suggest that Sumatran orangutans can now be added to the list of mammals which undergo a true menopause, which may ensure that females' final offspring can be reared to independence.

Comparative evaluation of point of care assay with ELISA techniques for quantifying serum concentrations of ustekinumab in inflammatory bowel disease patients

ObjectivesTo evaluate the analytical performance and clinical utility of the POC-AFIAS assay in comparison with two ELISA established assays for quantifying serum concentrations of ustekinumab. MethodsA prospective study was conducted. Consecutive serum samples from adult patients undergoing treatment with ustekinumab were collected. Three analytical techniques were compared for the quantification of ustekinumab serum concentrations: the AFIAS-10® POC assay (POC-AFIAS), the Promonitor® ELISA assay (ELISA-PRO), and the ELISA Ridascreen® assay (ELISA-RDSC). Ustekinumab concentrations were evaluated within three therapeutic ranges: <1μg/mL, 1–4.5μg/mL, and >4.5μg/mL. Statistical analysis included Pearson correlation, intra-class correlation coefficient, and Bland–Altman analysis. ResultsA total of 104 patients were included in the study. The median ustekinumab concentrations measured were 5.22μg/mL (POC-AFIAS), 3.99μg/mL (ELISA-PRO), and 4.50μg/mL (ELISA-RDSC). Strong correlations were observed between techniques (POC-AFIAS and ELISA-PRO: r=0.921, POC-AFIAS and ELISA-RDSC: r=0.940, ELISA-PRO and ELISA-RDSC: r=0.976). The Bland–Altman analysis revealed a bias difference of 1.81μg/mL between POC-AFIAS and ELISA-PRO, and 1.27μg/mL between POC-AFIAS and ELISA-RDSC. Agreement rates varied by therapeutic range, with the highest agreement observed within the therapeutic range (97.3%) and lower agreement for supra-therapeutic concentrations (74.6%). ConclusionThis study demonstrated that the POC-AFIAS assay provides comparable results to established ELISA techniques for quantifying serum concentrations of ustekinumab, particularly within the therapeutic range. The findings suggest that the POC-AFIAS assay offers a rapid and effective tool for managing ustekinumab therapy in clinical practice.

Association of anti-HMGCR antibodies of the IgM isotype with refractory immune-mediated necrotizing myopathy.

Anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) autoantibodies are one of the myositis-specific antibodies which is associated with immune-mediated necrotizing myopathy (IMNM). However, the relationship between anti-HMGCR isotypes and prognosis has not yet been fully investigated. This study was conducted to gain insight into the association between anti-HMGCR isotypes and clinical, and prognosis in IMNM patients who were positive for anti-HMGCR antibodies. Levels of anti-HMGCR isotypes (IgG, IgA and IgM) were assessed by enzyme-linked immunosorbent assay (ELISA) in 123 consecutive serum samples obtained from 71 patients who were positive for anti-HMGCR IgG at baseline. Disease activity was assessed by manual muscle testing (MMT) 8, Physician's Global Assessment (PGA) visual analog scale (VAS), and muscle VAS. Baseline anti-HMGCR IgG levels were correlated with PGA VAS (r = 0.24; p = 0.04), muscle VAS (r = 0.32; p < 0.01), and MMT8(r=-0.24; p = 0.04), and baseline anti-HMGCR IgM levels were positively correlated with PGA VAS (r = 0.27, p = 0.02), muscle VAS (r = 0.24, p = 0.04). Anti-HMGCR IgM positive patients had a lower age of onset [29(25,46) vs. 51(33,65), p = 0.006], and a higher proportion of neck weakness (63.5% vs. 34.6%, p = 0.031) compared with anti-HMGCR IgM negative patients. Longitudinal analysis showed that the changes in anti-HMGCR IgG levels were correlated with the changes in the PGA VAS (β = 3.830; p < 0.0001), muscle VAS (β = 2.893; p < 0.0001), MMT8 (β=-19.368; p < 0.0001), and creatine kinase (CK) levels (β = 3900.05, p < 0.0001). Anti-HMGCR IgM levels were weakly correlated with anti-HMGCR IgA levels at baseline (r = 0.33, p < 0.01), and the variations in anti-HMGCR IgA levels were correlated with the changes in anti-HMGCR IgM levels during follow-up (β = 0.885; p < 0.0001). There were more patients with anti-HMGCR IgM who showed a refractory course than those who were with anti-HMGCR IgM negative (polycyclic course: 40% vs. 25%; chronic continuous course: 46.7% vs. 20.5%, p = 0.018). In anti-HMGCR IgG-positive IMNM patients, the levels of anti-HMGCR IgG are associated with disease activity, and anti-HMGCR IgM is associated with refractory outcome and poor prognosis.

Cardiac rejection diagnosis using serum mRNA encoding proteins related to fibrotic process

Abstract Background Finding a standardized, non-invasive method of diagnosis of rejection is still a challenge in the field of heart transplantation. A novel alternative for allograft rejection diagnosis is the detection of biomarkers present in serum. Cardiac fibrosis appears to be present in patients who are experiencing cardiac allograft rejection. Thus, the process of fibrosis could offer a wide range of candidate molecules to explore as possible biomarkers. Purpose This study aimed to determine the expression of fibrosis-related coding mRNAs in serum of patient with cardiac rejection and evaluate the diagnostic capacity as potential biomarkers for the non-invasive detection of mild (grade 1R) and moderate/severe (grade ≥2R) cellular rejection. Methods We included consecutive serum samples from transplant recipients undergoing routine endomyocardial biopsies. Non-coding RNA sequencing analysis (Illumina HiSeq 2500) was performed on 40 samples, 28 diagnosed with acute cellular rejection (Grade 1R, n=16; and Grade ≥2R, n=12) and 12 samples without cardiac rejection. Results We detected 228 mRNA related to fibrosis in serum, which 38 presented differential expression in patients with cardiac rejection of Grade ≥2R. We obtained 16 molecules that showed excellent diagnostic ability in moderate-severe rejection (AUC &amp;gt; 0.800). We highlight RELB that was able to detect mild rejection (AUC=0.734, p&amp;lt;0.05). We stand out TNS1 (AUC=0.972), COL4A2 (AUC=0.958) and JAK1 (AUC=0.944), p&amp;lt;0.0001, for its relevant diagnostic capacity of acute cellular rejection. Conclusions We propose serum mRNA levels of genes encoding fibrosis related proteins as potential biomarkers of cardiac rejection. We highlight the role of RELB due to its excellent diagnostic capacity for acute cellular rejection correlating with the severity of the episode.

Combining Serum miR-144-3p and miR-652-3p as Potential Biomarkers for the Early Diagnosis and Stratification of Acute Cellular Rejection in Heart Transplantation Patients.

There is a dire need for specific, noninvasive biomarkers that can accurately detect cardiac acute cellular rejection (ACR) early. Previously, we described miR-144-3p as an excellent candidate for detecting grade ≥2R ACR. Now, we investigated the combination of miR-144-3p with miR-652-3p, other differentially expressed serum miRNA we previously described, to improve diagnostic accuracy mainly in mild rejection to avoid reaching severe stages. We selected miR-652-3p from a preliminary RNA-seq study to be validated by reverse transcription-quantitative polymerase chain reaction on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. We confirmed the miR-652-3p overexpression (P < 0.0001) and its capability to discriminate between patients with and without ACR of any grade (P < 0.0001). The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (P < 0.0001). This combination resulted in a diagnostic efficacy for 1R (area under the curve = 0.794) and ≥2R (area under the curve = 0.892; P < 0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950; P < 0.0001) and ≥2R (odds ratio of 14.289; P < 0.01). We demonstrated that an appropriate combination of blood-based biomarkers could exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at an early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades.

Pilot study shows suppression of mineralocorticoid precursors under high-dose glucocorticoid therapy in pediatric acute lymphoblastic leukemia.

Glucocorticoids represent a key element in the treatment of pediatric acute lymphoblastic leukemia (ALL) and lead to adrenal suppression. We aimed to assess the differential response profile of adrenal steroids in children with ALL during BFM (Berlin-Frankfurt-Münster) induction treatment. Therefore, we performed liquid chromatography tandem-mass spectrometry (LC-MS/MS)-based steroid profiling of up to seven consecutive leftover morning serum samples derived from 11 patients (pts) with ALL before (day 0) and during induction therapy at days 1-5, 6-12, 13-26, 27-29, 30-35 and 36-40. 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (11S), cortisol, 11-deoxycorticosterone (DOC), corticosterone and aldosterone were determined in parallel. Subsequently, steroid concentrations were normalized by multiples of median (MOM) to adequately consider pediatric age- and sex-specific reference ranges. MOM-cortisol and its precursors MOM-11S and MOM-17OHP were significantly suppressed by glucocorticoid treatment until day 29 (P < 8.06 × 10-10, P < 5.102 × 10-5, P < 0.0076, respectively). Cortisol recovered in one of four pts at days 27-29 and in two of five pts at days 36-40. Among the mineralocorticoids, corticosterone was significantly suppressed (P < 3.115 × 10-6). Aldosterone and DOC showed no significant changes when comparing day 0 to the treatment time points. However, two ALL patients with ICU treatment due to the sepsis showed significantly lower MOM-DOC (P = 0.006436) during that time and almost always the lowest aldosterone compared to all other time points. Suppression of mineralocorticoid precursors under high-dose glucocorticoid therapy suggests a functional cross talk of central glucocorticoid regulation and adrenal mineralocorticoid synthesis. Our data should stimulate prospective investigation to assess potential clinical relevance.

Testing for isotypes does not help differentiating rheumatoid arthritis from other rheumatoid factor positive diseases.

Rheumatoid factors (RFs) are useful for diagnosis and classification of rheumatoid arthritis (RA). Nephelometric and turbidimetric techniques, which detect total RF but do not reveal the antibody isotype, are common diagnostic methods in clinical routine. Given the recent development of isotype-specific immunoassays, the detection of IgG, IgM, and IgA RFs represents an interesting challenge. The aim of the study was to evaluate whether specific RF tests performed as a second step after traditional nephelometry could help differentiating RA from other RF-positive diseases. We tested 117 consecutive serum samples that were RF-positive at nephelometry (BNII nephelometric analyzer, Siemens) for IgA, IgG, and IgM RF isotypes by a fluoroimmunoenzymatic assay (FEIA) on the Phadia 250 instrument (ThermoFisher). Fifty-five subjects had RA and 62 presented non-RA diagnoses. Eighteen sera (15.4%) were positive only by nephelometry, two were positive only for IgA RF, and the remaining 97 sera were all positive for IgM RF isotype (with or without IgG and IgA RF). Positive findings did not correlate with RA or non-RA diagnosis. Spearman rho correlation coefficient between nephelometric total RF and IgM isotype was moderate (0.657), and weak between total RF and IgA (0.396) and IgG (0.360) isotypes. Despite its low specificity, measurement of total RF by nephelometry still seems to be the method that performs best. As IgM, IgA, and IgG RF isotypes showed only a moderate correlation with total RF measurement, their diagnostic use as a second level test remains controversial.

Impact of allele-specific anti–human leukocyte antigen class I antibodies on organ allocation

Technological advances in the field of histocompatibility have allowed us to define anti–human leukocyte antigen (HLA) antibody specificity at the allelic level. However, how allele-specific antibodies affect organ allocation is poorly studied. We examined allelic specificities of class I HLA antibodies in 6726 consecutive serum samples from 2953 transplant candidates and evaluated their impact on the corresponding crossmatch and organ allocation. Out of 17 class I HLA antigens represented by >1 allele in the LABScreen single antigen bead assay, 12 had potential allele-specific reactivity. Taking advantage of our unbiased cohort of deceased donor-candidate testing (123,135 complement-dependent cytotoxicity crossmatches between 2014 and 2017), we estimated that the presence of allele-specific antibody detected using a single antigen bead assay (median fluorescence intensity, >3000) against only the rare allele was a poor predictor of a positive complement-dependent cytotoxicity crossmatch, with a positive predictive value of 0% to 7%, compared with 52.5% in allele-concordant class I HLA antibodies against A or B locus antigens. Further, we confirmed allele-specific reactivity using flow crossmatch in 3 scenarios: A11:01/A11:02, A68:01/A68:02, and B44:02/B44:03. Our results suggest that allele-specific antibodies may unnecessarily exclude transplant candidates (up to 10%) from organ offers by overcalling unacceptable antigens; incorporation of selective reactivity pattern in allocation may promote precision matching and more equitable allocation.

Single and double vaccination against Lawsonia intracellularis in foals: Investigation of the humoral immune response following different vaccination protocols

SummaryBackgroundEquine proliferative enteropathy (EPE) is an enteric disease induced by the bacterium Lawsonia intracellularis that causes sporadically or endemically severe enteritis in foals.ObjectivesThe current study aimed to describe the kinetic of serum antibodies against Lawsonia intracellularis by Immuno‐Peroxidase‐Monolayer‐Assay (IPMA) before and after single or double intra‐rectal vaccination of foals with the attenuated live vaccine Enterisol Ileitis® in comparison to non‐vaccinated control foals. Additionally, we evaluated whether seroconversion after vaccination might provide information about the protection against EPE.Study designProspective cohort study.MethodsFive consecutive serum samples were evaluated by IPMA from all 210 foals: group 1 (single vaccination) = 70; group 2 (two vaccinations) = 69; group 3 (control group) = 71. The study period stretched from 2 weeks before to 6 weeks after the first vaccination.ResultsThe analysis showed 944 (89.9%) seronegative samples. 45 samples of group 1 (single vaccination), 56 samples of group 2 (two vaccinations) and five samples of the control group were seropositive during the study period. A total of 63/210 (30%) foals seroconverted during the study period. In foals vaccinated once (n = 70), 32 foals seroconverted, in group 2 (two vaccinations, n = 69) 28 foals seroconverted and in the control foals (n = 71), three foals seroconverted. The vaccinated foals showed significantly more often seroconversion than the control foals (p &lt; 0.0001). After the end of the study period, a total of six foals (two vaccinated foals, and four non‐vaccinated foals) were diagnosed with EPE.Main limitationsA longer study period might have added more information on the humoral response to vaccination.ConclusionsMeasurable Lawsonia intracellularis‐antibodies can be detected after intra‐rectal vaccination, but serological testing the time around the vaccination does not seem to be a reliable method to assess the efficacy of the vaccination.

Lack of comparability of immunoassays for rheumatoid factor isotypes.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by the presence of autoantibodies that are used for classification of the disease. Though routine diagnostics is commonly restricted to measuring rheumatoid factor (RF) and anti-citrullinated protein antibodies, detection of RF IgM, IgG and IgA isotypes, may increase the power of RA serodiagnosis by reducing the number of seronegative patients as well as provide prognostic information. The agglutination-based RF assays, such as nephelometry or turbidimetry, are unable to differentiate isotypes. We compared three different immunoassays used in current laboratory practice to detect RF isotypes. We tested 117 consecutive serum samples that were positive for total RF at nephelometry, from 55RA and 62non-RA subjects. IgA, IgG, and IgM isotypes of RF were tested by immunoenzymatic (ELISA, Technogenetics), fluoroenzymatic (FEIA, ThermoFisher) and chemiluminescence (CLIA, YHLO Biotech Co.) immunoassays. Diagnostic performance differed considerably between the assays, especially with regard to RF IgG isotype. Agreement among methods by Cohen's kappa ranged from 0.05 (RF IgG CLIA vs. FEIA) to 0.846 (RF IgM CLIA vs. FEIA). The poor agreement observed in this study indicates substantial lack of comparability among assays for RF isotypes. Harmonization of these tests requires further efforts before their measurement can be used in clinical practice.

Investigating iron intake in risk of progression from islet autoimmunity to type 1 diabetes: The diabetes autoimmunity study in the young.

Studies of the role of iron in the risk of type 1 diabetes (T1D) have been inconsistent. Given that iron generates reactive oxygen radicals, which can lead to oxidative damage and apoptosis in the beta cells of the pancreas, we examined whether iron intake was associated with the risk of progressing to T1D in individuals with islet autoimmunity (IA), the pre-clinical phase of T1D. DAISY is a prospective cohort following 2,547 children at increased risk for IA and progression to T1D. IA is defined as at least two consecutive serum samples positive for at least one autoantibody (insulin, GAD, IA-2, or ZnT8). We measured dietary intake at the time of IA seroconversion in 175 children with IA, and of these, 64 progressed to T1D. We used Cox regression to examine the association between energy-adjusted iron intake and progression to T1D, adjusting for HLA-DR3/4 genotype, race/ethnicity, age at seroconversion, presence of multiple autoantibodies at seroconversion, and multiple vitamin use. In addition, we tested whether this association was modified by vitamin C or calcium intake. In children with IA, high iron intake (as defined as above the 75th percentile, > 20.3 mg/day) was associated with decreased risk of progression to T1D compared to moderate iron intake (as defined by the middle 25-75th percentiles, 12.7-20.3 mg/day) (adjusted hazard ratio (HR): 0.35; 95% confidence interval (CI): 0.15, 0.79). The association between iron intake and T1D was not modified by vitamin C nor calcium intake. In a sensitivity analysis, the removal of six children who had been diagnosed with celiac disease prior to IA seroconversion did not affect this association. Higher iron intake at the time of IA seroconversion is associated with a lower risk of progression to T1D, independent of multivitamin supplement use. Further research that includes plasma biomarkers of iron status is needed to investigate the relationship between iron and the risk of T1D.

Non-invasive diagnosis of cardiac rejection using small non-coding RNAs

Abstract Background Endomyocardial biopsy is the standard technique for rejection surveillance in heart transplantation recipients and is an invasive method with important limitations and clearly associated with both a risk of procedural complications and serious long-term sequelae when performed repeatedly. All of these considerations highlight the need to identify non-invasive alternative methods capable of discriminating between the different grades of rejection and reducing the need for endomyocardial biopsy. Purpose We conducted a non-targeted transcriptomic study focused on identifying serum small non-coding RNAs, mainly aimed on the less studied types, to evaluate their diagnostic accuracy for detecting rejection episodes. Methods We included consecutive serum samples from transplant recipients undergoing routine endomyocardial biopsies. Non-coding RNA sequencing analysis (Illumina HiSeq 2500) was performed on 40 samples, 28 diagnosed with RCA (RCA Grade 1R, n=16; and RCA Grade ≥2R, n=12) and 12 samples without cardiac rejection. Results 636 non-coding RNAs were detected among the scaRNAs, snoRNAs, snRNAs and miscRNAs biotypes. But, neither piRNA nor siRNA biotypes were detected. Of all of them, 41 were altered in patients with rejection Grade ≥2R, finding no alteration in the scaRNAs biotype. Three molecules of miscRNA biotype showed the best results with excellent diagnostic capacity in moderate/severe rejection: RN7SL840P (AUC=0.938, p&amp;lt;0.01), Y_RNA [ENSG00000223302] (AUC=0.931, p&amp;lt;0.0001) and Y_RNA [ENSG00000202273] (AUC=0.903, p&amp;lt;0.001). In addition, RN7SKP5 showed a good capacity for the diagnosis of Grade ≥2R (AUC=0.806, p&amp;lt;0.05) and preserving the accuracy at Grade 1R (AUC=0.781, p&amp;lt;0.05). Conclusions The 6.5% of the small non-coding RNAs detected have differential expression in patients with acute cellular rejection. The non-detection of the piRNAs and siRNAs biotypes stands out, as well as the absence of alteration in the scaRNAs biotype. We propose serum miscRNA levels as potential biomarkers of cardiac rejection. We highlight the role of RN7SL840P due to its excellent diagnostic capacity for RCA, and RN7SKP5 since it also allows the detection of mild rejection. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Combining serum miR-144-3p and miR-652-3p as potential biomarkers for the early diagnosis and stratification of acute cellular rejection in heart transplantation patients

Abstract Background There is a dire need for specific, noninvasive biomarkers that can accurately and early detect cardiac acute cellular rejection (ACR). Previously, we described miR-144-3p as an excellent candidate to detect Grade ≥2R ACR. Purpose Now, we investigated the combination of miR-144-3p with other differentially expressed serum miRNA that we previously described to improve diagnostic accuracy, mainly in mild rejection to avoid reaching severe stages. Methods We selected miR-652-3p, miR-6747-3p and miR-221-3p from a preliminary RNA-seq study to be validated by RT-qPCR on 212 consecutive serum samples from transplantation recipients undergoing routine endomyocardial biopsies to subsequently combine them with miR-144-3p results and investigate their diagnostic capability. Results We confirmed the miR-652-3p overexpression (p&amp;lt;0.0001) and its capability to discriminate between patients with and without ACR of any grade (p&amp;lt;0.0001), but not miR-6747-3p or miR-221-3p. The combined serum levels of miR-144-3p and miR-652-3p were significantly higher in patients with rejection regardless of posttransplantation time (p&amp;lt;0.0001). This combination resulted in a diagnostic efficacy for 1R (AUC=0.794) and ≥2R (AUC=0.892) (p&amp;lt;0.0001) that was superior to each biomarker alone. Furthermore, it was a strong independent predictor of ACR for 1R (odds ratio of 10.950, p&amp;lt;0.0001) and ≥2R (odds ratio of 14.289, p&amp;lt;0.01). Conclusions We demonstrated that an appropriate combination of blood-based biomarkers can exhibit greater efficiency for cardiac rejection diagnosis. The combined detection of abnormal expression of miR-144-3p and miR-652-3p in the serum of ACR patients can improve the diagnostic sensitivity of rejection at the early stage and contribute to increasing the diagnostic accuracy, mainly in the lower rejection grades. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Hepatitis E Virus Prevalence and Associated Risk Factors in High-Risk Groups: A Cross-Sectional Study.

Seroepidemiology, risk factors to hepatitis E virus exposure, and prevalence of hepatitis E virus viremia have not yet been investigated among patients under immunosuppression or with liver disease that are high risk for infection in Turkey. In this cross-sectional study, 292 consecutive serum samples from renal transplant recipients, allogeneic hematopoietic stem cell transplant recipients, patients with acute hepatitis, and patients with chronic hepatitis C were prospectively collected in a ter- tiary university hospital. Sera were tested for hepatitis E virus immunoglobulin G/immunoglobulin M and hepatitis E virus ribonucleic acid using commercial enzyme-linked immunosorbent assay and in-house nested polymerase chain reaction with Sanger sequencing, respectively. Sociodemographic, clinical, laboratory data, and risk factors were collected using a questionnaire and hospital database. Multiple logistic regression analysis was employed to identify independent predictors for anti-hepatitis E virus seropositivity. Among all patients, only 2 patients (1 renal transplant recipient and 1 patient with acute hepatitis) were identified as having hepatitis E virus genotype 3 viremia. Hepatitis E virus viremia rate was 0.6% in whole group. These patients showed no signs of chronic hepatitis E virus infection for 6 months and were spontaneously seroconverted 6 months after enrollment. Anti-hepatitis E virus IgG was positive in 29 patients yielding a hepatitis E virus seroprevalence of 9.9%. Older age (adjusted odds ratio: 1.03, 95% CI, 1.00-1.06; P = .022) and eating undercooked meat (adjusted odds ratio: 3.11, 95% CI, 1.08-8.92; P = .034) were independent risk factors to anti- hepatitis E virus seropositivity in all patients. Similarly, multiple logistic regression analysis demonstrated that age (adjusted odds ratio: 1.03, 95% CI, 0.99-1.07, P = .058) and eating undercooked meat (adjusted odds ratio: 5.77, 95% CI, 1.49-22.25, P = .011) were indepen- dent risk factors for anti-hepatitis E virus IgG positivity in the liver disease subgroup consisting of acute hepatitis and chronic hepatitis C patients. The hepatitis E virus seroprevalence rate was high (9.9%), despite low viremia rate (0.6%) in high-risk patients. The emer- gence of hepatitis E virus genotype 3 might indicate a serious problem for these patients. Future investigations are needed to elucidate foodborne transmission routes of hepatitis E virus in Turkey.

Seroprevalence and dynamics of anti-SARS-CoV-2 antibodies: a longitudinal study based on patients with underlying diseases in Wuhan

BackgroundAssessing the humoral immunity of patients with underlying diseases after being infected with SARS-CoV-2 is essential for adopting effective prevention and control strategies. The purpose of this study is to analyze the seroprevalence of people with underlying diseases and the dynamic change features of anti-SARS-CoV-2 antibodies.MethodsWe selected 100 communities in Wuhan using the probability-proportional-to-size sampling method. From these 100 communities, we randomly selected households according to a list provided by the local government. Individuals who have lived in Wuhan for at least 14 days since December 2019 and were ≥ 40 years old were included. From April 9–13, 2020, community staff invited all selected individuals to the community healthcare center in batches by going door-to-door or telephone. All participants completed a standardized electronic questionnaire simultaneously. Finally, 5 ml of venous blood was collected from all participants. Blood samples were tested for the presence of pan-immunoglobulins, IgM, IgA, and IgG antibodies against SARS-CoV-2 nucleocapsid protein and neutralising antibodies were assessed. During the period June 11–13, 2020 and October 9–December 5, 2020, all family members of a positive family and matched negative families were followed up twice.ResultsThe seroprevalence of anti-SARS-CoV-2 antibodies in people with underlying diseases was 6.30% (95% CI [5.09–7.52]), and that of people without underlying diseases was 6.12% (95% CI [5.33–6.91]). A total of 313 people were positive for total antibodies at baseline, of which 97 had underlying disease. At the first follow-up, a total of 212 people were positive for total antibodies, of which 66 had underlying disease. At the second follow-up, a total of 238 people were positive for total antibodies, of which 68 had underlying disease. A total of 219 participants had three consecutive serum samples with positive total antibodies at baseline. The IgG titers decreased significantly with or without underlying diseases (P < 0.05) within the 9 months at least, while the neutralizing antibody titer remained stable. The titer of asymptomatic patients was lower than that of symptomatic patients (baseline, P = 0.032, second follow-up, P = 0.018) in the underlying diseases group.ConclusionOur research focused on the serological changes of people with and without underlying diseases in a state of single natural infection. Regardless of the underlying diseases, the IgG titer decreased significantly over time, while there was no significant difference in the decline rate of IgG between with and without underlying diseases. Moreover, the neutralizing antibody titer remained relatively stable within the 9 months at least.

The Diagnosis of Normocalcaemic Hyperparathyroidism is Strikingly Dissimilar Using Different Commercial Laboratory Assays.

We assessed the impact of intact parathyroid hormone (iPTH) and adjusted calcium analyses on Abbott, Roche and Siemens analytical platforms in the diagnosis of normocalcaemic primary hyperparathyroidism (NCPHPT). These assays are used by over 85% of clinical laboratories in the UK. Over five months, consecutive serum samples from outpatients with NCPHPT in the laboratory with Abbott assays were identified, aliquoted and stored at -80°C. Frozen aliquots were transported monthly to the other two laboratories. After thawing, samples were mixed and analysed immediately for calcium, albumin and iPTH in the laboratories with Abbott, Roche and Siemens analytical platforms. Adjusted calcium was calculated using the equation used in the respective laboratory. Diagnostic concordance of iPTH and adjusted calcium were assessed using manufacturer-provided assay-specific reference intervals and the pathology harmony reference interval respectively. Fifty-five patients with NCPHPT were identified using Abbott assays. Of these, 16 (29.1%) and 11 (20.0%) had NCPHPT, 9 (16.4%) and 13 (23.6%) had hypercalcaemic primary hyperparathyroidism, and 30 (54.6%) and 31 (56.4%) patients had normal results when analysed in laboratories with Roche and Siemens assays, respectively. The diagnosis of NCPHPT was strikingly different depending on the commercial assay used. There is a pressing need for iPTH assay harmonisation and robust reference intervals. Reference intervals may become invalid if an assay drifts, as exemplified by adjusted calcium in this study.

Helminth Infections in Children.

Helminth Infections in Children.

Longitudinal changes of serum protein N-Glycan levels for earlier detection of pancreatic cancer in high-risk individuals

BackgroundSurveillance of individuals at risk of developing pancreatic ductal adenocarcinoma (PDAC) has the potential to improve survival, yet early detection based on solely imaging modalities is challenging. We aimed to identify changes in serum glycosylation levels over time to earlier detect PDAC in high-risk individuals. MethodsIndividuals with a hereditary predisposition to develop PDAC were followed in two surveillance programs. Those, of which at least two consecutive serum samples were available, were included. Mass spectrometry analysis was performed to determine the total N-glycome for each consecutive sample. Potentially discriminating N-glycans were selected based on our previous cross-sectional analysis and relative abundances were calculated for each glycosylation feature. Results165 individuals (“FPC-cohort” N = 119; Leiden cohort N = 46) were included. In total, 97 (59%) individuals had a genetic predisposition (77 CDKN2A, 15 BRCA1/2, 5 STK11) and 68 (41%) a family history of PDAC without a known genetic predisposition (>10-fold increased risk of developing PDAC). From each individual, a median number of 3 serum samples (IQR 3) was collected.Ten individuals (6%) developed PDAC during 35 months of follow-up; nine (90%) of these patients carried a CDKN2A germline mutation. In PDAC cases, compared to all controls, glycosylation characteristics were increased (fucosylation, tri- and tetra-antennary structures, specific sialic linkage types), others decreased (complex-type diantennary and bisected glycans). The largest change over time was observed for tri-antennary fucosylated glycans, which were able to differentiate cases from controls with a specificity of 92%, sensitivity of 49% and accuracy of 90%. ConclusionSerum N-glycan monitoring may support early detection in a pancreas surveillance program.

Analysis of the concentration of selected serological parameters in patients undergoing antibiotic treatment of Lyme disease and assessment of their potential application in the control of the therapy effectiveness - pilot study.

The aim of the study was to analyze the concentrations of some serological parameters in patients undergoing antibiotic therapy for LD, and to assess the potential use of IgG anti-C6VlsE in the assessment of treatment effectiveness. The study involved 21 patients diagnosed with LD. To determine the levels of anti-C6VlsE IgG, metalloproteinases and cytokines, blood was collected three times: before treatment, immediately after treatment, and 3-4 months after treatment. Immediately after the antibiotic therapy, all the patients in the study group declared improvement in their health. At 3-4 months post-treatment, 50% of patients reported recurrence of the disease symptoms. The anti-C6VlsE IgG concentrations at the analyzed time points exhibited varied dynamics: in 15% of patients, a decrease in the concentration of anti-C6VlsE was observed in the three consecutive serum samples; an increase of anti-C6VlsE IgG was detected in 35% of cases; in 15% of the subjects, the concentration of anti-C6VlsE IgG increased immediately after the antibiotic therapy and declined after 3-4 months, whereas a reverse situation was noted in 10% of the patients. The presence of high concentrations of anti-C6VlsE IgG is associated with appearance of response to Borreliella spp. infection, which suggests that the parameter can be used in the diagnosis of the active form of borreliosis. However, from a practical point of view, a marker of successful elimination of infection should indicate the effectiveness or failure of antibiotic therapy within a relatively short post-treatment period. The anti-C6VlsE IgG antibodies do not seem to satisfy this provision.