Differential Detection of Five Mouse-Infecting Helicobacter Species by Multiplex PCR

Several species of helicobacter have been isolated from laboratory mice, including H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, which appear to be the most common. The most widely used published method for molecular detection of these agents is PCR amplification of a conserved region of 16S rRNA, but differential speciation requires restriction enzyme digestion of the amplicons. This study was undertaken to determine PCR conditions that would simultaneously and specifically identify each of the five common species without restriction enzyme analyses. First, we designed novel and specific PCR primers for H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, using sequences from the heterologous regions of 16S rRNA. Because of comigration of amplified products, we next identified P17, an H. bilis-specific protein; P25, an H. hepaticus-specific protein; and P30, an H. muridarum-specific protein by screening genomic DNA expression libraries of each species. Primers were designed from these three genes, plus newly designed, species-specific 16S rRNA primers for H. rodentium and H. typhlonius that could be utilized for a five-plex PCR. The sizes of the amplicons from H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius were 435, 705, 807, 191, and 122 bp, respectively, allowing simultaneous detection and effective discrimination among species.     

Recombinant Helicobacter bilis Protein P167 for Mouse Serodiagnosis in a Multiplex Microbead Assay

Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated to purified, H. bilis-specific, recombinant proteins P167C and P167D and bacterial membrane extracts from H. bilis and Helicobacter hepaticus. For detecting H. bilis infection in the microbead multiplex assay, P167C and P167D provided significantly higher sensitivities (94 and 100%, respectively) and specificities (100 and 95%, respectively) than membrane extract (78% sensitivity and 65% specificity). Microbead multiplex assay results were validated by enzyme-linked immunosorbent assay. Purified recombinant proteins showed low batch-to-batch variation; this feature allows for ease of quality control, assay robustness, and affordability. Thus, recombinant antigens are highly suitable in the multiplex microbead assay format for serodetection of H. bilis infection.     

Pattern of C3, iC3b, and C3d in patients hospitalized for acute asthma.

Twenty-three patients hospitalized for acute asthma were studied for a peripheral blood complement profile consisting of C3, C4, C3d, iC3b, C4d, and Bb concentrations. Compared with normals (n = 22) and patients (n = 10) with acute bacterial infections (ABI), asthmatic patients had significantly higher serum C3 concentrations (P less than .001). Plasma C3d levels and iC3b in asthmatic patients were both comparable to those observed in normal controls, whereas patients with ABI had significantly higher iC3b levels than both other groups. The ratio of iC3b to C3 concentrations were similar in asthmatic patients and controls, and iC3b levels were correlated with total serum C3 levels in asthmatic patients (r = .55, p less than .001) as well as in normal (r = .69, p less than .001). Both of these groups had significantly lower iC3b to C3 ratios compared with the ABI group (P less than .0001). Also observed in asthmatic patients were a significant correlation between serum C4 and C3 levels (r = .83, P less than .001) and a lower mean ratio of plasma C4d to C4 compared with normals (P less than .005). This profile of complement alterations is distinct from that observed in acute bacterial infection. These changes in asthmatic patients may relate to an acute phase reaction phenomenon affecting complement and/or complement regulatory proteins.     

Abbreviated measures of food sufficiency validly estimate the food security level of poor households: measuring household food security

This study was designed to develop an abbreviated method that captures both the qualitative and quantitative dimensions of household food security (HFS). Women in poor and very poor households (n = 238) in a peri-urban barrio in Caracas, Venezuela, provided data on food availability and their perception of food resource constraints and hunger experiences within the home. Socioeconomic data and food-related behavior that may predict HFS levels were gathered. On average, the top 12 food contributors of energy provided 81% and predicted more than 90% of the variation in households' total energy availability using stepwise regression analysis. On the other hand, a 4-point 12-item scale was shown to have face, content and construct validity with reiterative testing, factor analysis and a Chronbach's alpha coefficient of 0.92. Assessing predictors of energy availability together with a self-perceived HFS scale may provide a valid and reliable method for identifying and monitoring food security levels among poor urban households.     

Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing.     

Escore de Cálcio Coronário e Estratificação do Risco de Doença Arterial Coronariana em Pacientes com Acidente Vascular Encefálico Isquêmico Aterosclerótico e não-Aterosclerótico

BackgroundIschemic Stroke (IS) and Coronary Artery Disease (CAD) frequently coexist and share atherosclerotic disease risk factors. According to the American Heart Association, IS subtypes may be considered CAD risk equivalents, but the evidence for non-atherosclerotic IS is uncertain. Additionally, the Coronary Calcium Score (CCS) is an accurate marker to address CAD risk; however, CCS distribution between IS subtypes is not well characterized.ObjectivesTo compare the CCS between atherosclerotic and non-atherosclerotic IS groups; and to determine which covariates were associated with high CCS in IS.MethodsThis cross-sectional design included all patients with IS, 45 to 70 years of age at the time of the stroke, consecutively admitted to a rehabilitation hospital between August 2014 and December 2016, without prevalent CAD. All patients underwent CT scanning for CCS measurement. CCS≥100 was considered a high risk for CAD, with a significance level of p<0.05.ResultsFrom the 244 studied patients (mean age 58.4±6.8 years; 49% female), 164 (67%) had non-atherosclerotic etiology. The proportions of CCS≥100 were similar between the atherosclerotic and the non-atherosclerotic groups (33% [n=26] x 29% [n=47]; p= 0.54). Among all IS patients, only age ≥60 years was independently associated with CCS≥100 (OR 3.5; 95%CI 1.7-7.1), accounting for hypertension, dyslipidemia, diabetes, sedentarism, and family history of CAD.ConclusionAtherosclerotic IS did not present a greater risk of CAD when compared to non-atherosclerotic IS according to CCS. Only age ≥60 years, but not etiology, was independently associated with CCS≥100.     

Nutrient Intakes among Brazilian Children Need Improvement and Show Differences by Region and Socioeconomic Level

Brazil is the most populous country in South America. Using 24 h dietary data, we compared the nutrient intakes of 4–13-year-olds to reference values and tested for regional and socioeconomic (SES) differences. A considerable proportion reported intakes below the Estimated Average Requirements (EAR) for vitamins E (78.1%, 96.5%), D (100% for both), and calcium (80.5%, 97.7%) for 4–8 and 9–13-year-olds, respectively. Few exceeded Adequate Intakes (AI) for potassium or fiber. Older children reported greater inadequacies and, while there was regional variability, patterns of inadequacy and excess tended to be similar. For vitamin C, the percent of children below EAR in the Northeast and Southeast was lower than in the South. Most children, regardless of SES, had energy intakes within the Acceptable Macronutrient Distribution Ranges (AMDRs) for carbohydrates and protein. Over a quarter reported total energy from fat less than the AMDR, and inversely associated with SES (low 50.9%, moderate 26.0%, and high 15.0%), but also exceeding the percentage of energy recommendation for saturated fat, increasing with SES (low 18.1%, moderate 38.9%, and high 48.8%). The contrast observed between the diets of young Brazilians and recommendations underscores the need for individual and regional environmental interventions to promote healthier dietary patterns.