Multiplexed flow cytometric approach for detection of anti-SARS-CoV-2 IgG, IgM and IgA using beads covalently coupled to the nucleocapsid protein.

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we developed an innovative strategy for simultaneous detection of immunoglobulin G (IgG), IgM and IgA. The SARS-CoV-2 nucleocapsid protein was covalently bound to functional beads surface applying sulpho-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free assay efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensitivity of 88·5-96·2% and specificity of 100%. This novel strategy opens a new avenue for flow cytometry-based diagnosis.

Interleukin-18 (rs187238) and glucose transporter 4 (rs5435) polymorphisms in Euro-Brazilians with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease with a strong genetic component that has been associated with several genetic loci. Interleukin 18 (IL-18) is a potent proinflammatory cytokine, which is involved in the innate and adaptive immune responses, and in the pathogenesis of various diseases including T1D. Glucose transporter 4 (GLUT4) is known to be an insulin-responsive glucose transporter and has been associated with various diseases, including diabetes mellitus. We investigated the association of the polymorphisms rs187238 (IL-18) and rs5435 (GLUT4) in a case-control study in Euro-Brazilians with T1D (N = 136) and healthy subjects (N = 144). Real-time PCR with TaqMan® fluorescent probes were applied for genotyping. All polymorphisms were in Hardy-Weinberg equilibrium. The minor allele frequencies for the G-allele (rs187238; IL-18) in healthy and T1D groups were 28.5% [95%CI = 23-34%] vs 31.6% [95%CI = 26-37%], P = 0.416, and for the T-allele (rs5435, GLUT4) were 33% [95%CI = 28-39] vs 27% [95%CI = 23-33%], P = 0.167, respectively. Genotype comparisons for both polymorphisms showed no significant differences (P > 0.05). The polymorphisms rs187238 and rs5435 were not associated with T1D in the studied population. The minor allele frequencies for both polymorphisms were similar to those of other Caucasian populations.

Leptin (rs7799039) and solute carrier family 30 zinc transporter (rs13266634) polymorphisms in Euro-Brazilian pregnant women with gestational diabetes.

Leptin (LEP), a protein that plays a fundamental role in the metabolism of energy reserves, and the solute carrier family 30 A8 zinc transporter (SLC30A8) have been consistently associated with diabetes. Women with gestational diabetes are at moderate risk of developing diabetes type 1 and 2 after pregnancy, in addition to complications to the fetus. We investigated the association of the polymorphisms rs7799039 (LEP) and rs13266634 (SLC30A8) in a case-control study in Euro-Brazilians with gestational diabetes (GDM, N = 134) and healthy pregnant women (control, N = 180). Real-time PCR with fluorescent probes (TaqMan system) was applied to genotyping. All polymorphisms were in Hardy-Weinberg equilibrium. The minor allele frequencies, for healthy and GDM, respectively, for the A-allele (LEP gene rs7799039) were 40.3% (95%CI = 35-45%) vs 36.6% (95%CI = 31-42%), P = 0.345; and for the T-allele (SLC30A8 gene rs13266634) were 27.8% (95%CI = 23-32%) vs 23.5% (95%CI = 18-29%), P = 0.227. Genotype comparisons for both polymorphisms showed no significant difference (P > 0.05). The polymorphisms rs7799039 and rs13266634 were not associated with GDM in the population studied (P > 0.05). The minor allele frequencies for both polymorphisms were similar to those of other Caucasian populations.

Polymorphism E23K (rs5219) in the KCNJ11 gene in Euro-Brazilian subjects with type 1 and 2 diabetes.

Insulin secretion is regulated by ATP-sensitive potassium channels (KATP). The potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11) gene, located on chromosome 11p15.1, encodes the subunit Kir6.2 that forms the pore region of KATP channels in pancreatic ß-cells. Among the single nucleotide polymorphisms (SNPs) associated with KCNJ11, the E23K polymorphism (rs5219) promotes a substitution (G > A) of a glutamic acid residue for lysine at position 23. The E23K SNP has been associated with diabetes in several populations, although with controversial results. The aim of this study was to evaluate the association of the E23K SNP with type 1 and 2 diabetes in a case-control study approved by the Ethics Committee. We genotyped 458 Euro-Brazilian individuals, classified as healthy (control group, CTRL, N = 217), patients with type 1 diabetes mellitus (T1D, N = 102), and patients with type 2 diabetes mellitus (T2D, N = 139). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using BanII restriction digestion. The restriction fragments were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The genotype (EE/EK/KK) frequencies (%) for the CTRL group (38.2/50.2/11.6), T1D (34.3/52.0/13.7), and T2D (38.2/48.9/12.9) were in Hardy-Weinberg equilibrium and there were no significant differences (CRTL vs T1D, P = 0.771; CRTL vs T2D, P = 0.937; T1D vs T2D, P = 0.831). The minor allele frequencies (MAF; K) for CTRL (37.0%), T1D (39.7%), and T2D (37.4%) were not different among the groups (P > 0.05). The MAF value for healthy subjects was similar to other Caucasian populations (34.5-37.5%). In summary, the E23K polymorphism (rs5219) was not associated with type 1 or 2 diabetes mellitus in the studied population.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories.

The mass profiles of cell-free extracts of 180 commensal and pathogenic strains of Escherichia coli were determined by MALDI-TOF mass spectrometry (MS). While some peaks were highly conserved in all E. coli, several peaks occurred only in some strains, showing heterogeneity among them. We did not detect strain-specific peaks for any of the E. coli categories tested. However, review of the fully conserved and the variable peaks suggested that MALDI-TOF MS has the potential to distinguish commensal and uropathogenic E. coli strains. Additionally, eight Shigella sonnei isolates were tested and found to be indistinguishable from E. coli by MALDI-TOF MS under the test conditions.

Proteins differentially expressed by Shiga toxin-producing Escherichia coli strain M03 due to the biliar salt sodium deoxycholate.

Shiga toxin-producing Escherichia coli (STEC) can cause conditions ranging from diarrhea to potentially fatal hemolytic uremic syndrome. Enteropathogen adaptation to the intestinal environment is necessary for the development of infection, and response to bile is an essential characteristic. We evaluated the response of STEC strain M03 to the bile salt sodium deoxycholate through proteomic analysis. Cell extracts of strain M03 grown with and without sodium deoxycholate were analyzed by two-dimensional electrophoresis; the differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Three proteins were found to be differentially expressed due to sodium deoxycholate. Glycerol dehydrogenase and phosphate acetyltransferase, which are involved in carbon metabolism and have been associated with virulence in some bacteria, were downregulated. The elongation factor Tu (TufA) was upregulated. This protein participates in the translation process and also has chaperone activities. These findings help us understand strategies for bacterial survival under these conditions.

Brand of dipotassium EDTA vacuum tube as a new source of pre-analytical variability in routine haematology testing.

This study assesses the use of different dry K2 (dipotassium) EDTA vacuum tubes and whether or not they might represent a bias in haematological testing. Blood was collected in three dipotassium EDTA vacuum tubes from different manufacturers: Venosafe, Vacuette and Vacutainer. Samples were analysed on an Advia 2120i analyser. Significant differences among results and biases were compared with current quality specifications. Significant differences were found for haematocrit (HCT), mean corpuscular volume (MCV), white blood cell count (WBC) and platelet distribution width (PDW) when comparing Venosafe vs. Vacuette; for MCV, WBC and PDW when comparing Venosafe vs. Vacutainer; and for HCT and MCV when comparing Vacuette vs. Vacutainer. Clinically significant variations were observed for HCT and PDW in Venosafe vs. Vacuette; PDW in Venosafe vs. Vacutainer; and HCT and MCV in Vacuette vs. Vacutainer. The use of dipotassium EDTA vacuum tubes from different manufacturers represent a clinically relevant source of variation for HCT, MCV and PDW.

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control.

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.

Polymorphisms of the promoter and exon 3 of the receptor for advanced glycation end products (RAGE) in Euro- and Afro-Brazilians.

The receptor for advanced glycation end products (RAGE or AGER), a member of the immunoglobulin superfamily, is involved in pathologies such as atherosclerosis and diabetes. Over 50 SNPs were reported for RAGE, among which were the promoter region polymorphisms -429T>C (rs1800625), -374T>A (rs1800624) and a 63-bp deletion (-407 to -345 bp), all related to increased RAGE expression. Additionally, in the exon 3, a putative site of binding ligands, the missense variation G82S (rs2070600) was associated with skin disorders in patients with diabetes. We have determined allele, genotype and haplotype frequencies of RAGE polymorphisms -429T>C, -374T>A, 63-bp deletion and G82S in Euro-Brazilians (n = 108) and Afro-Brazilians (n = 91), characterized according to the predominant ancestry of the individuals. The allele frequencies for Euro- and Afro-Brazilians were as follows: -429C, 12.5% vs. 12.1% (P = 0.90); -374A, 31.5% vs. 26.2% (P = 0.25); 63del, 0.0% vs. 3.8% (P = 0.004); and 82S, 1.9% vs. 0.6% (P = 0.24). Absolute linkage disequilibrium was found between the promoter polymorphisms -429T>C and -374T>A plus the 63-bp deletion (D'=1.000; P < 0.0001). The haplotype frequencies differed (P = 0.003) between Euro- and Afro-Brazilians. Our results showed that the frequencies of the 63-bp deletion were higher in Afro-Brazilians, while the other analysed polymorphisms were similarly distributed in the studied populations. The -374T>A plus 63-bp deletion polymorphism captures more than 80% of the haplotypic variation in the studied population.

Virulence characteristics and antimicrobial susceptibility of uropathogenic Escherichia coli strains.

Eight virulence factors associated with uropathogenic Escherichia coli (UPEC) were investigated in 204 clinical isolates of E. coli recovered from urine cultures at counts ≥10(5). The bacteria were classified into two groups according to the number of leukocytes in urine samples from which they were isolated: group I ≤8 leukocytes/hpf, 104 strains; group II >8 leukocytes/hpf, 100 strains. Two multiplex PCR systems were used to detect genes encoding adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and traT associated with serum resistance. The PAI marker for the virulence island identified in strains CFT072 and CVD432, a marker of enteroaggregative E. coli, was also investigated using PCR. The susceptibility profile of E. coli strains was determined by disk diffusion method. Ninety percent UPEC showed at least one of the virulence genes, the prevalence being traT (76%), aer (41%), PAI (32%), sfa (26%), pap (25%), cnf1 (18%), afa (6%), and hly (5%). There was no significant difference in the distribution of virulence genes between groups I and II. A significantly higher degree of virulence was detected in UPEC group II. The CVD432 gene was not detected in any of the UPECs. Fifty-nine percent of the strains were resistant to at least one of the antimicrobials that we tested; the most common being resistance to ampicillin (51%) and trimethoprim-sulfamethoxazole (44%).

Transillumination: a new tool to eliminate the impact of venous stasis during the procedure for the collection of diagnostic blood specimens for routine haematological testing.

INTRODUCTION: The collection of diagnostic blood specimens for routine haematological testing (RHT) is traditionally performed with tourniquet. However, the transillumination devices based on cold near-infrared LEDs have been formerly proposed as a valuable tool for identifying reliable venous accesses, especially in patients with difficult or small veins, such as children. This study was aimed to evaluate whether a transillumination device can advantageously replace the use of the tourniquet during the procedure for collection of blood specimens for RHT and thereby eliminating the discomfort and risk of spurious results caused by excessive or prolonged venous stasis. METHODS: Two hundred and fifty volunteers were divided into five groups (G1, G2, G3, G4 and G5) to compare the results of RHT between blood sample collected with transilluminator device (left arm) and with tourniquet application (right arm) for 30 s(G1), 60 s(G2), 90 s(G3), 120 s(G4) and 180 s(G5). RESULTS: No significant increases were observed in any of the haematological parameters tested in G1 when compared with blood collected by the transilluminator device. From G2 to G5, significant increases were observed for the platelet count, red blood cell count, haemoglobin, haematocrit, white blood cell count, neutrophils, monocytes and eosinophils. From G3-G5, further increases were observed for lymphocytes. Clinically significant variations were, however, observed for basophils in G2; red blood cell count, haemoglobin, haematocrit and basophils in G3 and eosinophils in G3 only. CONCLUSION: As such, considering that inappropriate use of the tourniquet is commonplace, we conclude that transillumination devices can represent a suitable tool to eliminate the venous stasis and to improve the quality of phlebotomy procedures.

The functional polymorphisms -429T>C and -374T>A of the RAGE gene promoter are not associated with gestational diabetes in Euro-Brazilians.

The receptor for advanced glycation end products (RAGE or AGER) is a multiligand member of the immunoglobulin superfamily. RAGE is expressed in several tissues, including human myometrium, chorionic villi and placenta. Advanced glycation end products are the best studied ligands of RAGE; they have pro-inflammatory actions in human gestational tissues, increasing oxidative stress and the release of cytokines and prostaglandins. We investigated the association of RAGE gene promoter polymorphisms -429T>C (rs1800625) and -374T>A (rs1800624) with gestational diabetes. A sample of 750 unrelated European origin pregnant Brazilian women were classified as nondiabetic (control group, N = 600) or having gestational diabetes (N = 150) according to American Diabetes Association 2009 criteria. Genotyping was performed by PCR-RFLP. The frequencies of the rare alleles -429C (6.3 versus 9.1%) and -374A (26 versus 30%) were not significantly different between the gestational diabetes patients and healthy pregnant women. Also, the -429T>C and -374T>A polymorphisms were not associated with body mass index, lipid profile, fasting glycemia, HbA1C, or insulin requirement. We found that functional promoter polymorphisms of the RAGE gene were not associated with gestational diabetes or its complications in these Euro-Brazilian patients.

Butyrylcholinesterase activity and risk factors for coronary artery disease.

The aim of this study was to verify which risk factors for coronary artery disease (CAD) are independently correlated with butyrylcholinesterase (BChE) activity. We studied 88 White individuals (43 males) aged 47.3+/-15.7 years (mean+/-SD; range: 14.0-80.0 years) including 38 with hyperlipidemia, 30 with hypertension and 5 with diabetes mellitus (DM). Simple correlation analysis showed that BChE activity was positively correlated with age, sex, body mass index, hypertension and DM, as well as with triglycerides (TGs), total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B (Apo B). However, after a step-wise multiple regression analysis, the only risk factors for CAD that showed independent correlations with BChE activity were, in descending order of importance, Apo B, TGs and DM. Our findings seem to reinforce suggested associations of BChE activity with lipoprotein synthesis and with hypertension, as well as supporting previous data on the relation of BChE activity with disturbances found in diabetes mellitus.

An improved method for butyrylcholinesterase phenotyping.

An improved method for the identification of butyrylcholinesterase phenotypes is proposed. It is based on modifications of a method that uses alpha-naphthyl acetate as substrate and DL-propranolol and Ro2-0683 as inhibitors. The proposed modifications make the method more rapid and increase the accuracy of the determinations of the phenotypes tested (BCHE U, BCHE UF, BCHE UA, BCHE AK, BCHE AF, and BCHE A). These modifications make the method even more adequate for population studies and clinical routine.

The C5 isozyme of serum cholinesterase and adult weight.

The relationship between the CHE2 locus of serum cholinesterase (BChE) and adult human weight was studied in a sample of 225 CHE2 C5+ individuals and 225 CHE2 C5- controls matched for sex, height, age and race. With respect to the intensity of the C5 band staining (scored 1-6), 113 individuals had faint C5 bands (scores 1-3) and 112 intense C5 bands (scores 4-6). The individuals with intense CHE2 C5+ phenotype showed a significantly lower mean adult weight (64.66 +/- 0.73 kg) when compared to their controls (70.59 +/- 0.97 kg) and a significant reduction in weight variance (59.81 and 105.18, respectively). Individuals with faint C5 bands, although showing a negative correlation between weight and C5 band intensity, did not differ from their controls in mean weight.

Frequency of the CHE1*K allele of serum cholinesterase in a sample from southern Brazil.

The frequency of the CHE1*K allele was estimated as 2.04 +/- 2.02% in a population sample from Southern Brazil. Previously reported estimates refer to the British population and are significantly higher than the present one. Our hypothesis is that the British frequencies may represent overestimates due to ascertainment conditions.