Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Study of Orally Disintegrating Tablets Using Erythritol as an Excipient Produced by Moisture-Activated Dry Granulation (MADG)

Moisture-activated dry granulation (MADG) is an eco-friendly granulation method that uses a small amount of water and insoluble excipients to absorb moisture. MADG is expected to improve productivity and reduce costs. Erythritol, an excipient used for preparing orally disintegrating tablets (ODTs), has poor tabletability and is difficult to form into tablets by conventional methods, such as high-shear granulation (HSG) and direct compression. In this study, we optimized the manufacturing conditions for ODTs to improve the tabletability of erythritol using MADG. The disintegration time of tablets made using the MADG method was approximately one-tenth that of those made using the HSG method, and the hardness was approximately 1.4 times higher. Moreover, MADG could delay disintegration and improve tabletability. We further attempted to optimize the manufacturing conditions using MADG, particularly in terms of the amount of water used. The disintegration time increased as the amount of added water increased. Moreover, water absorption tests revealed that capillary wetting decreased as the amount of water added increased, but the initial wetting did not change. These results suggested that the disintegration time was prolonged because of the increase in granule density and decrease in capillary wetting with the increase in the amount of added water. The hardness of the tablets increased because of the easy deformation of the granules after the addition of up to 3% water; however, when more than 3% water was added, the hardness decreased because of the aggregation of the granules with the excess water. Finally, two-dimensional maps of the effect of the amount of added water and water activity indicated that tablets with a hardness of ≥80 N and a disintegration time of ≤15 s could be produced by adjusting the amount of added water to within the range of 2.2–3.3% and water activity to 0.3–0.53. These results indicate that MADG can improve the tabletability of erythritol and be used for the granulation of ODTs. Tablets with appropriate hardness and disintegration properties can be produced by adjusting the water content to approximately 2.7% and the water activity to approximately 0.4 when producing ODTs with MADG

Long-term colonization exceeding six years from early infancy of Bifidobacterium longum subsp. longum in human gut

Abstract Background The importance of the gut microbiota at the early stage of life and their longitudinal effect on host health have recently been well investigated. In particular, Bifidobacterium longum subsp. longum, a common component of infant gut microbiota, appears in the gut shortly after birth and can be detected there throughout an individual’s lifespan. However, it remains unclear whether this species colonizes in the gut over the long term from early infancy. Here, we investigated the long-term colonization of B. longum subsp. longum by comparing the genotypes of isolates obtained at different time points from individual subjects. Strains were isolated over time from the feces of 12 subjects followed from early infancy (the first six months of life) up to childhood (approximately six years of age). We also considered whether the strains were transmitted from their mothers’ perinatal samples (prenatal feces and postnatal breast milk). Results Intra-species diversity of B. longum subsp. longum was observed in some subjects’ fecal samples collected in early infancy and childhood, as well as in the prenatal fecal samples of their mothers. Among the highlighted strains, several were confirmed to colonize and persist in single individuals from as early as 90 days of age for more than six years; these were classified as long-term colonizers. One of the long-term colonizers was also detected from the corresponding mother’s postnatal breast milk. Quantitative polymerase chain reaction data suggested that these long-term colonizers persisted in the subjects’ gut despite the existence of the other predominant species of Bifidobacterium. Conclusions Our results showed that several strains belonging to B. longum subsp. longum colonized in the human gut from early infancy through more than six years, confirming the existence of long-term colonizers from this period. Moreover, the results suggested that these strains persisted in the subjects’ gut while co-existing with the other predominant bifidobacterial species. Our findings also suggested the importance of microbial-strain colonization in early infancy relative to their succession and showed the possibility that probiotics targeting infants might have longitudinal effects. Trial Registration TRN: ISRCTN25216339. Date of registration: 11/03/2016. Prospectively registered

Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic <i>Clostridium difficile</i> in Human Stools

<div><p>Background</p><p><i>Clostridium difficile</i> is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of <i>C. difficile</i> infection. A sensitive and accurate detection method of <i>C. difficile</i>, especially toxigenic strains is indispensable for the epidemiological investigation.</p><p>Methods</p><p>TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, <i>tcdB</i>, and <i>tcdA</i> genes of <i>C. difficile</i> was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of <i>C. difficile</i>. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by <i>C. difficile</i> selective culture (CDSC).</p><p>Results</p><p>The analysis of <i>C. difficile</i>-spiked stools confirmed that qPCR quantified whole <i>C. difficile</i> (TcdA⁺TcdB⁺, TcdA⁻TcdB⁺, and TcdA⁻TcdB⁻ types), TcdB-producing strains (TcdA⁺TcdB⁺ and TcdA⁻TcdB⁺ types), and TcdA-producing strains (TcdA⁺TcdB⁺ type), respectively, with a lower detection limit of 10³ cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were <i>C. difficile</i>-positive by qPCR: TcdA⁺TcdB⁺ strain in six specimens and TcdA⁻TcdB⁻ strain in the other six. CDSC detected <i>C. difficile</i> in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole <i>C. difficile</i> and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of <i>C. difficile</i> detected was 10^4.5 cells/g of stool, suggesting that <i>C. difficile</i> constituted a very small fraction of intestinal microbiota.</p><p>Conclusion</p><p>Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of <i>C. difficile</i>.</p></div

Longitudinal investigation of carriage rates, counts, and genotypes of toxigenic Clostridium difficile in early infancy

Asymptomatic infant carriers of toxigenic Clostridium difficile are suggested to play a role in the transmission of C. difficile infection (CDI) in adults. However, the mode of C. difficile carriage in infants remains to be fully elucidated. We investigated longitudinal changes in carriage rates, counts, and strain types of toxigenic C. difficile in infants. Stools collected from 111 healthy infants in Belgium periodically from birth until the age of 6 months were examined by quantitative PCR targeting 16S rRNA and toxin genes. Toxigenic C. difficile was detected in 18 of 111 infants (16%) in the period up to the age of 6 months. The carriage rate of toxigenic C. difficile remained below 5% until the age of 3 months. The carriage rate increased to 13% 1 week after weaning (average age, 143 days) and reached 16% at the age of 6 months. Counts of toxigenic C. difficile bacteria ranged from 104 to 108 cells/g of stool. Notably, two infants retained >108 cells/g of stool for at least several weeks. Average counts in the 18 infants hovered around 107 cells/g of stool from the age of 3 days until the age of 6 months, showing no age-related trend. Genotyping of toxigenic C. difficile isolates from the 18 infants revealed that 11 infants each retained a particular monophyletic strain for at least a month. The genotype most frequently identified was the same as that frequently identified in symptomatic adult CDI patients. Thus, toxigenic C. difficile strains-potential causes of CDI in adults-colonized the infants' intestines.</p

Comparison of detection results of toxigenic <i>C. difficile</i> or toxins by qPCR, <i>C. difficile</i> selective culture (CDSC), and enzyme immunoassay (EIA).

a<p>Toxigenic types were identified on the basis of qPCR counts for the three genes, according to the criteria described in Materials and Methods.</p>b<p>The toxigenic type of isolates was determined on the basis of PCR amplification of <i>tcdA</i> and <i>tcdB</i> by using the method of Kato <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111684#pone.0111684-Kato1" target="_blank">[15]</a>.</p>c<p>na, not applicable.</p><p>Comparison of detection results of toxigenic <i>C. difficile</i> or toxins by qPCR, <i>C. difficile</i> selective culture (CDSC), and enzyme immunoassay (EIA).</p

Comparison of qPCR analytical curves among <i>C. difficile</i> strains.

a<p>Each analytical curve of different <i>C. difficile</i> strains was generated with serial dilutions ranging from 10 to 10⁵ cells per reaction. X-axis is bacterial cells applied to the reaction (log₁₀ cells/reaction) and Y-axis is the <i>C_T</i> values obtained.</p>b<p>Differences in <i>C_T</i> values compared with that of the type strain (DSM 1296^T) are indicated.</p><p>Comparison of qPCR analytical curves among <i>C. difficile</i> strains.</p

Comparison of detection results of <i>C. difficile</i> between qPCR and <i>C. difficile</i> selective culture (CDSC).

a<p>“<i>C. difficile</i> positive/negative” was defined by presence/absence of qPCR amplification with the 16S rRNA primers-probe set.</p>b<p>“<i>C. difficile</i> positive/negative” was defined by presence/absence of <i>C. difficile</i> isolation by means of stool culture.</p><p>Comparison of detection results of <i>C. difficile</i> between qPCR and <i>C. difficile</i> selective culture (CDSC).</p

<i>C. difficile</i> carriage rates in four nursing home populations.

<p>On the basis of the qPCR counts for three genes (16S rRNA, <i>tcdA</i>, and <i>tcdB</i>), the toxigenic types (A⁺B⁺, A⁻B⁺, or A⁻B⁻) of <i>C. difficile</i> predominating in individual specimens were identified. The rates of carriage of each toxigenic type of <i>C. difficile</i> at three stool samplings (S1, S2, and S3) were calculated with respect to each nursing home (n = 11, 14, 24, and 33, respectively) and the total population (n = 82).</p