Rapid and Sensitive Detection of an Intracellular Pathogen in Human Peripheral Leukocytes with Hybridizing Magnetic Relaxation Nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample’s magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen

Corporate reporting and disclosures in the emerging capital market of Kuwait:the perceptions of users and preparers

The objective of this paper is to investigate the perceptions of users and preparers regarding financial disclosure practices in annual reports of Kuwaiti listed firms. To measure participants' views, a questionnaire survey was distributed in Kuwait between October and December 2012, to preparers (financial managers) and users (financial analysts) within Kuwaiti listed companies. The study compares between the perceptions of financial managers and financial analysts regarding disclosing information in corporate annual reports as well as the main obstacles facing the disclosure process and what the problems restricting the use of companies' annual reports. The study also seeks to investigate whether there is a perceived need for improving the usefulness of Kuwaiti companies' annual reports for decision-making. The results, based on 137 responses, indicate that accounting practices in Kuwaiti firms are firmly rooted in a decision-usefulness tradition with management and the board of directors viewed as the key audience for reporting information. Indeed, the annual reports of Kuwaiti listed companies are perceived as the most important sources of information. On the whole both users and preparers shared similar concerns regarding the volume of information contained within annual reports; however, their views differed in terms of identifying potential solutions. The results of the study are likely to have implications for decision makers, the academic community and accounting standard setters. 2018 Macmillan Publishers Ltd., part of Springer Nature

Cytokine expression in subjects with Mycobacterium avium ssp. paratuberculosis positive blood cultures and a meta-analysis of cytokine expression in Crohn's disease.

OBJECTIVES: 1) Culture Mycobacterium avium ssp. paratuberculosis (MAP)from blood, 2) assess infection persistence, 3) determine Crohn's disease (CD) cytokine expression, 4) compare CD cytokine expression to tuberculosis, and 5) perform a meta-analysis of cytokine expression in CD. METHODS: The Temple University/Abilene Christian University (TU/ACU) study had a prospective case control design with 201 subjects including 61 CD patients and 140 non-CD controls. The culture methods included MGIT, TiKa and Pozzato broths, and were deemed MAP positive, if IS900 PCR positive. A phage amplification assay was also performed to detect MAP. Cytokine analysis of the TU/ACU samples was performed using Simple Plex cytokine reagents on the Ella ELISA system. Statistical analyses were done after log transformation using the R software package. The meta-analysis combined three studies. RESULTS: Most subjects had MAP positive blood cultures by one or more methods in 3 laboratories. In our cytokine study comparing CD to non-CD controls, IL-17, IFNγ and TNFα were significantly increased in CD, but IL-2, IL-5, IL-10 and GM-CSF were not increased. In the meta-analysis, IL-6, IL-8 and IL-12 were significantly increased in the CD patients. CONCLUSION: Most subjects in our sample had MAP infection and 8 of 9 subjects remained MAP positive one year later indicating persistent infection. While not identical, cytokine expression patterns in MAP culture positive CD patients in the TU/ACU study showed similarities (increased IL-17, IFNγ and TNFα) to patterns of patients with Tuberculosis in other studies, indicating the possibilities of similar mechanisms of pathogen infection and potential strategies for treatment

Genetic adult lactase persistence is associated with risk of Crohn's Disease in a New Zealand population

Background: Mycobacterium avium subspecies paratuberculosis (MAP) is an infective agent found in ruminants and milk products, which has been suggested to increase the risk of gastrointestinal inflammation in genetically susceptible hosts. It is hypothesized that lactase persistence facilitates exposure to such milk products increasing the likelihood of adverse outcomes. Individuals either homozygous or heterozygous for the T allele of DNA variant, rs4988235, located 14kb upstream from the LCT locus, are associated with having lactase persistence. The aim of this study was to determine whether lactase persistence as evident by the T allele of rs4988235 is associated with Crohn's Disease (CD) in a New Zealand population. Findings: Individuals homozygous for the T allele (T/T genotype) showed a significantly increased risk of having CD as compared with those homozygous for the C allele (OR = 1.61, 95% CI = 1.03-2.51). Additionally, a significant increase in the frequency of the T allele was observed in CD patients (OR = 1.30, 95% CI = 1.05-1.61, p = 0.013), indicating that the T allele encoding lactase persistence was associated with an increased risk of CD. Conclusions: Our findings indicate that lactase persistence as evident by the presence of the T allele of rs4988235 is associated with risk of CD in this New Zealand Caucasian population

Identification and Characterisation of Pseudomonas 16S Ribosomal DNA from Ileal Biopsies of Children with Crohn's Disease

Molecular analysis of bacterial 16S rRNA genes has made a significant contribution to the identification and characterisation of bacterial flora in the human gut. In particular, this methodology has helped characterise bacterial families implicated in the aetiology of inflammatory bowel disease (IBD). In this study we have used a genus specific bacterial 16S PCR to investigate the prevalence and diversity of Pseudomonas species derived from the ileum of children with Crohn's disease (CD), and from control children with non-inflammatory bowel disease (non-IBD) undergoing their initial endoscopic examination. Fifty eight percent of CD patients (18/32) were positive using the Pseudomonas PCR, while significantly fewer children in the non-IBD group, 33% (12/36), were PCR positive for Pseudomonas (p<0.05, Fischer's exact test). Pseudomonas specific 16S PCR products from 13 CD and 12 non-IBD children were cloned and sequenced. Five hundred and eighty one sequences were generated and used for the comparative analysis of Pseudomonas diversity between CD and non-IBD patients. Pseudomonas species were less diverse in CD patients compared with non-IBD patients. In particular P.aeruginosa was only identified in non-IBD patients

On the Action of Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis

BACKGROUND: Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. METHODOLOGY: The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (% −ΔcGI). PRINCIPAL FINDINGS: The positive control antibiotic (clarithromycin) has ≥85% −ΔcGI at a concentration of 0.5 µg/ml. The negative control (ampicillin) has minimal inhibition at 64 µg/ml. MAP ATCC 19698 shows ≥80% −ΔcGI for both agents by 4 µg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate. CONCLUSIONS: We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % −ΔcGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups

Groundwater Chemistry and Blood Pressure: A Cross-Sectional Study in Bangladesh

Background: We assessed the association of groundwater chemicals with systolic blood pressure (SBP) and diastolic blood pressure (DBP). Methods: Blood pressure data for ≥35-year-olds were from the Bangladesh Demographic and Health Survey in 2011. Groundwater chemicals in 3534 well water samples from Bangladesh were measured by the British Geological Survey (BGS) in 1998–1999. Participants who reported groundwater as their primary source of drinking water were assigned chemical measures from the nearest BGS well. Survey-adjusted linear regression methods were used to assess the association of each groundwater chemical with the log-transformed blood pressure of the participants. Models were adjusted for age, sex, body mass index, smoking status, geographical region, household wealth, rural or urban residence, and educational attainment, and further adjusted for all other groundwater chemicals. Results: One standard deviation (SD) increase in groundwater magnesium was associated with a 0.992 (95% confidence interval (CI): 0.986, 0.998) geometric mean ratio (GMR) of SBP and a 0.991 (95% CI: 0.985, 0.996) GMR of DBP when adjusted for covariates except groundwater chemicals. When additionally adjusted for groundwater chemicals, one SD increase in groundwater magnesium was associated with a 0.984 (95% CI: 0.972, 0.997) GMR of SBP and a 0.990 (95% CI: 0.979, 1.000) GMR of DBP. However, associations were attenuated following Bonferroni-correction for multiple chemical comparisons in the full-adjusted model. Groundwater concentrations of calcium, potassium, silicon, sulfate, barium, zinc, manganese, and iron were not associated with SBP or DBP in the full-adjusted models. Conclusions: Groundwater magnesium had a weak association with lower SBP and DBP of the participants

On the Action of Cyclosporine A, Rapamycin and Tacrolimus on M. avium Including Subspecies paratuberculosis

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) may be zoonotic. Recently the "immuno-modulators" methotrexate, azathioprine and 6-MP and the "anti-inflammatory" 5-ASA have been shown to inhibit MAP growth in vitro. We concluded that their most plausible mechanism of action is as antiMAP antibiotics. The "immunosuppressants" Cyclosporine A, Rapamycin and Tacrolimus (FK 506) treat a variety of "autoimmune" and "inflammatory" diseases. Rapamycin and Tacrolimus are macrolides. We hypothesized that their mode of action may simply be to inhibit MAP growth. METHODOLOGY: The effect on radiometric MAP (14)CO(2) growth kinetics of Cyclosporine A, Rapamycin and Tacrolimus on MAP cultured from humans (Dominic & UCF 4) or ruminants (ATCC 19698 & 303) and M. avium subspecies avium (ATCC 25291 & 101) are presented as "percent decrease in cumulative GI" (%-DeltacGI.) PRINCIPAL FINDINGS: The positive control clofazimine has 99%-DeltacGI at 0.5 microg/ml (Dominic). Phthalimide, a negative control has no dose dependent inhibition on any strain. Against MAP there is dose dependent inhibition by the immunosuppressants. Cyclosporine has 97%-DeltacGI by 32 microg/ml (Dominic), Rapamycin has 74%-DeltacGI by 64 microg/ml (UCF 4) and Tacrolimus 43%-DeltacGI by 64 microg/ml (UCF 4) CONCLUSIONS: We show heretofore-undescribed inhibition of MAP growth in vitro by "immunosuppressants;" the cyclic undecapeptide Cyclosporine A, and the macrolides Rapamycin and Tacrolimus. These data are compatible with our thesis that, unknowingly, the medical profession has been treating MAP infections since 1942 when 5-ASA and subsequently azathioprine, 6-MP and methotrexate were introduced in the therapy of some "autoimmune" and "inflammatory" diseases