Methicillin-resistant Staphylococcus aureus colonisation: epidemiological and molecular characteristics in an acute-care tertiary hospital in Singapore.

Current knowledge of methicillin-resistant Staphylococcus aureus (MRSA) colonisation in relation to epidemiological characteristics is incomplete. We conducted a cross-sectional study at an acute-care tertiary infectious diseases hospital of MRSA isolates identified through routine surveillance from January 2009 to December 2011. We randomly selected 205 MRSA isolates (119 inpatients) from 798 isolates (427 inpatients) for molecular profiling using multilocus sequence typing. Multilevel multinomial logistic regression was used to estimate odds ratio (OR) assessing the predilection of MRSA strains for anatomic sites, and associations of strains with human immunodeficiency virus (HIV) infection. The most frequent sequence types (STs) were 239, 22 and 45. The proportion of ST22 increased over the sampling period, replacing ST239 as the dominant lineage. However, ST239 remained the most prevalent among HIV-seropositive individuals who were six times more likely to be colonised with this strain than non-HIV patients (adjusted OR (aOR) 6.44, 95% confidence interval (CI) 1.94-21.36). ST45 was >24 times more likely to be associated with perianal colonisation than in the nares, axillae and groin sites (aOR 24.20, 95% CI 1.45-403.26). This study underlines the clonal replacement of MRSA in Singapore as previously reported but revealed, in addition, key strain differences between HIV-infected and non-infected individuals hospitalised in the same environment.

Epiregulin (EREG) variation is associated with susceptibility to tuberculosis.

Although host genetics influences susceptibility to Mycobacterium tuberculosis, the human genes regulating pathogenesis remain largely unknown. We used M. tuberculosis-stimulated macrophage gene expression profiling in conjunction with a case-control genetic association study to discover epiregulin (EREG), as a novel candidate tuberculosis (TB) susceptibility gene. Using a genome-wide association study dataset, we found that among the 21 genes with greater than 50-fold induction, EREG had the most polymorphisms associated with TB. We genotyped haplotype-tagging polymorphisms in discovery (N = 337 cases, N = 380 controls) and validation (N = 332 cases) datasets and an EREG polymorphism (rs7675690) was associated with susceptibility to TB (genotypic comparison; corrected P = 0.00007). rs7675690 was also associated more strongly with infections caused by the Beijing lineage of M. tuberculosis when compared with non-Beijing strains (controls vs Beijing, OR 7.81, P = 8.7 × 10(-5); non-Beijing, OR 3.13, P = 0.074). Furthermore, EREG expression was induced in monocytes and peripheral blood mononuclear cells stimulated with M. tuberculosis as well as TLR4 and TLR2/1/6 ligands. In murine macrophages, EREG expression induced by M. tuberculosis was MYD88- and TLR2-dependent. Together, these data provide the first evidence for an important role for EREG as a susceptibility gene for human TB.

Multilocus sequence typing subtypes of poultry Clostridium perfringens isolates demonstrate disease niche partitioning.

Clostridium perfringens is a ubiquitous and versatile pathogenic bacterium and is implicated in the etiology of the poultry diseases necrotic enteritis (NE) and poultry gangrene (PG). In this study, multilocus sequence typing was used to investigate genotypic relationships among 139 C. perfringens isolates from 74 flocks. These isolates had multiple disease, host, and environmental origins. The results indicated a polymorphic yet highly clonal population, with 79.6% of all isolates partitioning into one of six clonal complexes or two dominant sequence types, ST-9 and ST-31. The most prolific clonal complex, CC-1, contained 27.3% of all isolates and was not clearly associated with one particular disease. The subtypes CC-4 and ST-31 were highly associated with NE and represented 9.4% and 7.2% of the total isolates, respectively. No PG-associated and NE-associated C. perfringens isolates shared the same sequence type or clonal complex. NE-associated subtypes were more clonal and appeared more evolutionarily divergent than PG-associated subtypes, which tended to cluster in the more ancestral lineages alongside isolates from asymptomatic chickens and turkeys. Toxin gene screening identified cpb2 throughout these isolates and correlated the presence of netB with NE pathology. Previous investigations into the genetic basis of C. perfringens pathogenicity have focused on toxins and other variable genetic elements. This study presents the first sequence-based comparison of C. perfringens isolates recovered in clinical cases of PG and NE and demonstrates that niche specialization is observable in the core genomes of poultry-associated C. perfringens isolates, a concept with both epidemiological and evolutionary significance.

Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis.

Coronary occlusive disease is the leading cause of death in industrial nations and affects one in four adults. Although heart attacks are caused by occlusion of a coronary artery, some patients have occlusions without infarction because they have sufficient collateral vessels providing an alternate pathway for blood supply. Vascular endothelial growth factor (VEGF) is an angiogenic peptide that can stimulate collateral vessel development in the ischaemic myocardium. We used magnetic resonance imaging (MRI) and image processing to identify and quantify non-invasively the benefits related to VEGF infusion on collateral development in the heart. This was accomplished as a placebo-controlled study in the porcine model of chronic ischaemia that most closely mimics the human pathophysiology of progressive coronary occlusion. Image series converted to a space-time map demonstrated that with treatment the ischaemic zone was smaller and the contrast arrival delay was less, which resulted in better ejection fraction and regional wall thickening. These findings demonstrate in a manner applicable to humans, that VEGF improves collateral blood supply, resulting in improved cardiac global and regional function after and in spite of coronary artery occlusion.

New genetic associations detected in a host response study to hepatitis B vaccine.

The immune response to hepatitis B vaccination differs greatly among individuals, with 5-10% of healthy people failing to produce protective levels of antibodies. Several factors have been implicated in determining this response, chiefly individual genetic variation and age. Aiming to identify genes involved in the response to hepatitis B vaccination, a two-stage investigation of 6091 single-nucleotide polymorphisms (SNPs) in 914 immune genes was performed in an Indonesian cohort of 981 individuals showing normal levels of anti-HBs versus 665 individuals displaying undetectable levels of anti-HBs 18 months after initial dose of the vaccine. Of 275 SNPs identified in the first stage (476 normal/372 nonresponders) with P<0.05, significant associations were replicated for 25 polymorphisms in 15 genes (503 normal/295 nonresponders). We validated previous findings (HLA-DRA, rs5000563, P-value combined=5.57 x 10(-10); OR (95%CI)=0.61 (0.52-0.71)). In addition, we detected a new association outside of the human leukocyte antigen loci region that passed correction for multiple testing. This SNP is in the 3' downstream region of FOXP1, a transcription factor involved in B-cell development (P-value combined=9.2 x 10(-6); OR (95%CI)=1.38 (1.2-1.6)).These findings might help to understand the biological reasons behind vaccine failure and other aspects of variation in the immune responses of healthy individuals.

What is the evidence of a role for host genetics in susceptibility to influenza A/H5N1?

The apparent family clustering of avian influenza A/H5N1 has led several groups to postulate the existence of a host genetic influence on susceptibility to A/H5N1, yet the role of host factors on the risk of A/H5N1 disease has received remarkably little attention compared to the efforts focused on viral factors. We examined the epidemiological patterns of human A/H5N1 cases, their possible explanations, and the plausibility of a host genetic effect on susceptibility to A/H5N1 infection. The preponderance of familial clustering of cases and the relative lack of non-familial clusters, the occurrence of related cases separated by time and place, and the paucity of cases in some highly exposed groups such as poultry cullers, are consistent with a host genetic effect. Animal models support the biological plausibility of genetic susceptibility to A/H5N1. Although the evidence is circumstantial, host genetic factors are a parsimonious explanation for the unusual epidemiology of human A/H5N1 cases and warrant further investigation.

Phosphate release and force generation in skeletal muscle fibers.

Rapid laser pulse-induced photolysis of an adenosine triphosphate precursor in muscle fibers abruptly initiated cycling of the cross-bridges. The accompanying changes in tension and stiffness were related to elementary mechanochemical events of the energy-transducing mechanism. When inorganic phosphate was present at millimolar concentrations during liberation of adenosine triphosphate in the absence of calcium, relaxation was accelerated. Steady active tension in the presence of calcium was decreased but the approach to final tension was more rapid. These results suggest that, during energy transduction, formation of the dominant force-generating cross-bridge state is coupled to release of inorganic phosphate in a reaction that is readily reversible.

Polymorphism in the 5'-end of the aldose reductase gene is strongly associated with the development of diabetic nephropathy in type I diabetes.

Recent studies suggest that the gene encoding aldose reductase (ALR2), the enzyme that converts glucose to sorbitol, may confer susceptibility to microvascular disease. DNA from 275 British Caucasian patients with type I diabetes and 102 normal healthy control patients were typed for a (CA)n dinucleotide repeat polymorphic marker in the 5'-region of the ALR2 gene using polymorase chain reaction (PCR). A highly significant decrease in the frequency of the Z+2 allele was found in patients with nephropathy (nephropathy group) compared with those with no complications after a 20-year duration of diabetes (uncomplicated group) (12.7 vs. 38.2%, respectively, chi2 = 18.6, P < 0.00001); this was accompanied by an increase in the Z-2 allele in the nephropathy group (32.0 vs. 12.7% in the uncomplicated group). The nephropathy group also had a significant decrease in the Z/Z+2 genotype compared with the uncomplicated patients (10.7 vs. 44.7%, chi2 = 16.0, P < 0.0001) and an increased frequency of the Z/Z-2 genotype. There was no significant association with diabetic retinopathy. These results demonstrate that the ALR2 gene may play a role in susceptibility to diabetic nephropathy; individuals with the Z+2 allele are more than seven times less likely to develop diabetic renal disease than those without this marker. This marker may prove valuable in screening for patients with diabetic nephropathy at diagnosis of diabetes.

Importance of imaging method over imaging modality in noninvasive determination of left ventricular volumes and ejection fraction: assessment by two- and three-dimensional echocardiography and magnetic resonance imaging.

OBJECTIVES: This study sought to determine the concordance between biplane and volumetric echocardiography and magnetic resonance imaging (MRI) strategies and their impact on the classification of patients according to left ventricular (LV) ejection fraction (EF) (LVEF). BACKGROUND: Transthoracic echocardiography and MRI are noninvasive imaging modalities well suited for serial evaluation of LV volume and LVEF. Despite the accuracy and reproducibility of volumetric methods, quantitative biplane methods are commonly used, as they minimize both scanning and analysis times. METHODS: Thirty-five adult subjects, including 25 patients with dilated cardiomyopathies, were evaluated by biplane and volumetric (cardiac short-axis stack) cine MRI and by biplane and volumetric (three-dimensional) transthoracic echocardiography. Left ventricular volume, LVEF and LV function categories (LVEF > or =55%, >35% to <55% and < or =35%) were then determined. RESULTS: Biplane echocardiography underestimated LV volume with respect to the other three strategies (p < 0.01). There were no significant differences (p > 0.05) between any of the strategies for quantitative LVEF. Volumetric MRI and volumetric echocardiography differed by a single functional category for 2 patients (8%). Six to 11 patients (24% to 44%) differed when comparing biplane and volumetric methods. Ten patients (40%) changed their functional status when biplane MRI and biplane echocardiography were compared; this comparison also revealed the greatest mean absolute difference in estimates of EF for those subjects whose EF functional category had changed. CONCLUSIONS: Volumetric MRI and volumetric echocardiographic measures of LV volume and LVEF agree well and give similar results when used to stratify patients with dilated cardiomyopathy according to systolic function. Agreement is poor between biplane and volumetric methods and worse between biplane methods, which assigned 40% of patients to different categories according to LVEF. The choice of imaging method (volumetric or biplane) has a greater impact on the results than does the choice of imaging modality (echocardiography or MRI) when measuring LV volume and systolic function.

Randomised clinical trial: safety, tolerability, pharmacokinetics and pharmacodynamics of repeated doses of TAK-438 (vonoprazan), a novel potassium-competitive acid blocker, in healthy male subjects.

BACKGROUND: TAK-438 (vonoprazan) is a potassium-competitive acid blocker that reversibly inhibits gastric H(+) , K(+) -ATPase. AIM: To evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of TAK-438 in healthy Japanese and non-Japanese men. METHODS: In two Phase I, randomised, double-blind, placebo-controlled studies, healthy men (Japan N = 60; UK N = 48) received TAK-438 10-40 mg once daily at a fixed dose level for 7 consecutive days. Assessments included safety, tolerability, pharmacokinetics and pharmacodynamics (intragastric pH). RESULTS: Plasma concentration-time profiles of TAK-438 at all dose levels showed rapid absorption (median Tmax ≤2 h). Mean elimination half-life was up to 9 h. Exposure was slightly greater than dose proportional, with no apparent time-dependent inhibition of metabolism. There was no important difference between the two studies in AUC0-tau on Day 7. TAK-438 caused dose-dependent acid suppression. On Day 7, mean 24-h intragastric pH>4 holding time ratio (HTR) with 40 mg TAK-438 was 100% (Japan) and 93.2% (UK), and mean night-time pH>4 HTR was 100% (Japan) and 90.4% (UK). TAK-438 was well tolerated. The frequency of adverse events was similar at all dose levels and there were no serious adverse events. There were no important increases in serum alanine transaminase activity. Serum gastrin and pepsinogen I and II concentrations increased with TAK-438 dose. CONCLUSIONS: TAK-438 in multiple rising oral dose levels of 10-40 mg once daily for 7 days was safe and well tolerated in healthy men and caused rapid, profound and sustained suppression of gastric acid secretion throughout each 24-h dosing interval. Clinicaltrials.gov identifiers: NCT02123953 and NCT02141711.

X-linked recessive congenital muscle fiber hypotrophy with central nuclei: abnormalities of growth and adenylate cyclase in muscle tissue cultures.

Muscle cells in cultures established from biopsy specimens of two children with an infantile-fatal form of X-linked recessive muscle fiber smallness with central nuclei showed an unusual ability to proliferate through numerous passages. Ultrastructurally, the cultured muscle fibers appeared very immature even after several weeks. The nuclei were large, the number of ribosomes was greatly increased, the myofibrils remained unstriated, and glycogen was accumulated in large lakes. The plasmalemma bound concanavalin A, alpha-bungarotoxin, and ruthenium red normally, but with tannic acid it did not show the dark binding of mature fibers. Biochemically, in the cultured muscle fibers, beta-adrenergic receptors were quantitatively normal. The level of adenylate cyclase in membranes was less than in cultured normal muscle; this defect could be responsible for impaired control mechanisms resulting in the other abnormalities observed.

Relation between the number of image planes and the accuracy of three-dimensional echocardiography for measuring left ventricular volumes and ejection fraction.

The relation between accuracy of 3-dimensional echocardiography (3DE) in determining left ventricular end-diastolic volume, end-systolic volume, and ejection fraction (compared with magnetic resonance imaging) and the number of component planes used for 3DE ventricular reconstruction was evaluated in 41 adult subjects with normal (n = 24) and abnormal (n = 17) left ventricles. Accuracy and confidence of 3DE gradually increased with use of additional component planes, so that > or = 10 planes from both parasternal and apical windows provided 3DE reconstructions that accurately predict magnetic resonance imaging-measured left ventricular volumes and ejection fraction with confidence.

Effect of increased body mass index on accuracy of two-dimensional echocardiography for measurement of left ventricular volume, ejection fraction, and mass.

We used 2- and 3-dimensional echocardiography to determine left ventricular volume, mass, and ejection fraction in overweight (body mass index [BMI] > or = 25 kg/m2), obese (BMI > or = 30 kg/m2), and control (BMI < 25 kg/m2) subjects. Compared with corresponding magnetic resonance imaging measurements, 3-dimensional echocardiography is more accurate than 2-dimensional echocardiography in all patients, but particularly in overweight and obese subjects.

Analysis of the peripheral T-cell receptor V beta repertoire in newly diagnosed patients with type I diabetes.

Type I diabetes is an autoimmune disease characterised by a marked activation of peripheral T cells around the time of clinical diagnosis. Studies of T-cell antigen receptor V beta (TCRBV) gene usage in type I diabetes have been conflicting. Using a semi-quantitative polymerase chain reaction technique and flow cytometry we have investigated the TCRBV gene usage of 13 newly diagnosed patients with type I diabetes and 11 normal healthy controls. No preferential TCRBV gene usage was found between patients and controls even after matching for HLA-DR3 and/or -DR4. In addition, no significant differences in TCRBV gene usage were found between sequential samples taken over a period of up to 7 months following diagnosis. These results suggest that the TCR repertoire of these patients is heterogeneous and it is unlikely that a single 'pathogenic' T-cell clone is dominant at the clinical onset of the disease.

Effect of the Factor V Leiden mutation on the severity of meningococcal disease.

BACKGROUND: One of the most serious complications of meningococcal disease is the syndrome of purpura fulminans, which is characterized by intravascular thrombosis and hemorrhagic infarction of skin, limbs and digits. The reasons why some patients with meningococcal disease develop purpura fulminans while others have minimal thrombotic and skin involvement despite having profound septic shock are not yet understood. The Factor V Leiden mutation (FV(L)) is associated with thrombotic events, and we hypothesized that children carrying FV(L) who develop meningococcal disease may be at increased risk of purpura fulminans. METHODS: We determined the FV(L) genotype by PCR and restriction enzyme digestion (Mnl1) in 259 children with meningococcal disease and 80 healthy controls. In addition 79 parents of children with fatal meningococcal disease were studied. RESULTS: There was no significant increase in the frequency of FV(L) in patients with meningococcal disease (10%) as compared with healthy controls (9%) or with the parents of children who died of meningococcal disease (12%). Although the mortality was not increased in patients heterozygous for FV(L), they had increased complications of purpura fulminans, as assessed by requirement for skin grafting, referral to plastic surgeon and/or amputation. Among survivors 5 of 24 (21%) of those heterozygous for FV(L) had complications, compared with 14 of 233 (7%) who were wild type [P < 0.03; relative risk, 3.1 (95% confidence intervals, 1.2 to 7.9)]. CONCLUSIONS: FV(L) exacerbates purpura fulminans in meningococcal disease but does not have a significant effect on mortality.

Impact of on-line endocardial border detection on determination of left ventricular volume and ejection fraction by transthoracic 3-dimensional echocardiography.

This study was performed to determine whether use of on-line automated border detection (ABD) could reduce data analysis time for 3-dimensional echocardiography (3DE) while maintaining accuracy of 3DE in measures of left ventricular (LV) volumes and ejection fraction (EF). The study proceeded in 2 phases. In the validation phase, 20 subjects were examined with the use of 3DE and of monoplane 2-dimensional (2D) ABD. Results were compared with the reference standard of magnetic resonance imaging (MRI). In the test phase, 20 subjects underwent two 3DE studies (once with images optimized for visual border definition and once with images optimized for ABD border tracking) and a conventionally used 2D ABD study. For 3DE, volumes and EF were determined with the use of manually traced borders and ABD. Analysis times were recorded with a digital stopwatch. In the validation phase, 3DE and MRI results correlated very well (r = 0.99) without systematic differences. Comparison of 2D ABD with MRI showed good correlation for LV volumes (r >/= 0.90) and EF (r = 0.85) despite significant underestimation. For the test phase, Acoustic Quantification-optimized 3-dimensional datasets underestimated end-diastolic volume and EF relative to visually optimized 3-dimensional datasets regardless of whether borders were hand-traced or ABD was used. However, correlations ranged from r = 0.96 to r = 0.98 for LV volumes and 0.88 to 0.91 for LV EF and were superior to those for 2D ABD. Data analysis times decreased moderately with the use of ABD, but scan times increased; total study times were unchanged. Use of on-line ABD with 3DE reduces data analysis time and is more accurate than conventional monoplane 2D ABD but results in underestimation of LV volumes and EF. Additional automated postprocessing techniques may be required to obtain accurate measures, consistently using 3DE in conjunction with on-line ABD.

Three-dimensional echocardiographic reconstruction of right ventricular volume: in vitro comparison with two-dimensional methods.

Two-dimensional echocardiographic measures of right ventricular volume are limited by the asymmetric and crescentic shape of that ventricle and the difficulty in obtaining standardized views. We have developed a three-dimensional echocardiographic system that automatically integrates images and positional data and calculates right ventricular volume without the need for geometric assumptions or standardized views and a surfacing algorithm that takes advantage of the full three-dimensional data set. The accuracy of this system was studied and compared with two-dimensional methods in 12 gel-filled excised human right ventricles (18 to 74 ml). Volumes calculated by three-dimensional echocardiography correlated well with actual values (r = 0.99) and agreed more closely with them than did those obtained by two-dimensional methods (p < 0.02).

Mitoxantrone combined with vincristine, cyclophosphamide and fluorouracil for advanced breast cancer. A study of short-term response rate.

50 patients with advanced breast cancer were treated with the combination of Mitoxantrone 10mg/m2 IV day 2, 5-Fluorouracil 400mg/m2 IV and Cyclophosphamide 300mg/m2 IV day 3, 4, 5, 6 of each monthly cycle. 49 patients are evaluable for toxicity and 47 for efficacy after three months of treatment. Hematologic toxicity was substantial and dose-limiting, with one toxic death early in the trial. Other toxicities were moderate and manageable in this short-term study. The response rate after three cycles was 53% +/- 14% with 4 complete remissions, 21 partial remissions, 16 stable disease and 6 progressions. Using the fixed response rate hypothesis of Gehan generalised by Lee and Wesley, with an expected response rate of 60% consistent with the reported response rate of advanced breast cancer to Adriamycin containing regimens, we conclude that the combination studied is not less efficient for the induction of remissions in advanced breast cancer than comparable combinations with Adriamycin. As there is now substantial experimental and clinical evidence of reduced toxicity, mainly on the cardiac muscle, of Mitoxantrone as compared to Adriamycin, we feel that the routine substitution of the latter by the former in chemotherapy for advanced breast cancer is justifiable.

Chromosome 7q35 and susceptibility to diabetic microvascular complications.

Aldose reductase (ALR2), the first enzyme of the polyol pathway, may plan an important role in the pathogenesis of diabetic microvascular complications. The gene coding for ALR2 has been localized to chromosome 7q35. Using an ALR2 probe in conjunction with the restriction endonuclease Bam-HI, we have investigated the ALR2 locus of 128 patients with type I diabetes. A significant decrease in the frequency of the 8.2 kilobase (kb) Bam-HI ALR2 genotype and 8.2 kb allele was found in patients with nephropathy (nephropaths) compared to those with retinopathy alone (retinopaths) (p < 0.05 and 0.25, respectively). We have previously shown that an RFLP of the T-cell antigen receptor constant beta-chain (TCRBC) locus, which is also localized to chromosome 7q35, is strongly associated with susceptibility to microvascular complications. The 128 patients were genotyped using the restriction endonuclease Bgl-II and a TCRBC probe. The 10/9.2-8.2 kb TCRBC-ALR2 genotype was significantly decreased in the nephropaths compared to the retinopaths (13.7% versus 43.6%, chi 2 = 10.1, p < 0.0025). The 10/9.2 and 9.2/9.2 kb TCRBC-ALR2 haplotypes were increased in the nephropaths compared to the retinopaths (32.5% versus 8.9% chi 2 = 10.9, p < 0.001). These results suggest that chromosome 7q35 harbors a gene(s) that is involved in the pathogenesis of microvascular complications. Interestingly, the gene coding for endothelial nitric oxide synthase has recently been localized to the same chromosomal region as ALR2.

The angiotensin I-converting enzyme (ACE) locus is strongly associated with age and duration of diabetes in patients with type I diabetes.

We have investigated the frequency of the angiotensin I-converting enzyme (ACE) insertion/ deletion (I/D) polymorphism in 249 patients with type I diabetes and 162 normal healthy controls. There was no significant difference in the frequency of ACE genotypes between those patients with diabetic nephropathy (n = 72) (nephropaths) compared to those with no proteinuria after 20 years duration of diabetes (n = 86) (normoalbuminurics). There was, however, a significant difference in the frequency of ACE genotypes between the short-duration and long-term normoalbuminuric group (chi = 11.5, p = 0.001). Analysis of the ACE genotypes with respect to age and duration of diabetes showed that homozygosity for the insertion (I/I) genotype was significantly decreased with longer duration of diabetes (r2 = 92.7%, p < 0.009). No association was found with age in the normal controls. In conclusion, these results suggest that the ACE locus may be associated with longevity and survival in patients with type I diabetes rather than diabetic nephropathy or microvascular disease per se.