Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma,Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

Background

Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate.

Methods

We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples.

Results

Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR.

Conclusions

We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.     

Effects of Nutritional Supplementation on Fatigue,and Autonomic and Immune Dysfunction in Patients with End-Stage Renal Disease: A Randomized,Double-Blind,Placebo-Controlled,Multicenter Trial

Background

Fatigue is a predictor of cardiovascular events in patients with end-stage renal disease (ESRD) undergoing hemodialysis treatment. We hypothesized that multinutritional support would improve quality of life, fatigue symptoms, and potential quantitative measures including endocrine, immune and autonomic functions in patients with ESRD undergoing hemodialysis.

Methods

Two hundred and two hemodialysis patients were randomly assigned to receive active treatment (containing vitamin B1, vitamin B2, niacin, vitamin B6, vitamin B12, folic acid, vitamin C, carnitine, coenzyme Q10, naïve galacto-oligosaccharide, and zinc) or placebo after each dialysis session for 12 weeks. The patients and attending physicians were blinded to the treatment, and 172 patients (86 in each group) completed the study. Fatigue was evaluated via fatigue questionnaire at 0, 4, and 12 weeks. To assess human herpes virus (HHV) 6 and 7 reactivation, numbers of viral DNA copies were determined in saliva by polymerase chain reaction at weeks 0 and 12. Autonomic function was determined via measurement of beat-to-beat variation by using acceleration plethysmography.

Results

Clinical characteristics, changes in fatigue, quality of life score, endocrine functions, and laboratory data did not differ significantly between the two groups. Several parameters of heart rate variability significantly increased after nutritional treatment compared to placebo. Nutritional drink for 12 weeks significantly suppressed HHV7 DNA copy numbers. Similarly, HHV6 DNA copy numbers tended to be decreased by treatment but without reaching statistical significance.

Conclusions

Nutritional supplementation may modulate immune and autonomic dysfunction in ESRD patients undergoing hemodialysis.     

Association between Mouth Breathing and Atopic Dermatitis in Japanese Children 2–6 years Old: A Population-Based Cross-Sectional Study

As mouth breathing is associated with asthma and otitis media, it may be associated with other diseases. Therefore, this population-based cross-sectional study evaluated the association of mouth breathing with the prevalences of various diseases in children. Preschool children older than 2 years were included. A questionnaire was given to parents/guardians at 13 nurseries in Tokushima City. There were 468 valid responses (45.2%). We defined a subject as a mouth breather in daytime (MBD) if they had 2 or more positive items among the 3 following items: “breathes with mouth ordinarily,” “mouth is open ordinarily,” and “mouth is open when chewing.” We defined subjects as mouth breathers during sleep (MBS) if they had 2 or more positive items among the following 3 items: “snoring,” “mouth is open during sleeping,” and “mouth is dry when your child gets up.” The prevalences of MBD and MBS were 35.5% and 45.9%, respectively. There were significant associations between MBD and atopic dermatitis (odds ratio [OR]: 2.4, 95% confidence interval [CI]: 1.4–4.2), MBS and atopic dermatitis (OR: 2.4, 95% CI: 1.3–4.2), and MBD and asthma (OR: 2.2, 95% CI: 1.2–4.0). After adjusting for history of asthma and allergic rhinitis; family history of atopic dermatitis, asthma, and allergic rhinitis; and nasal congestion; both MBD (OR: 2.6, 95% CI: 1.3–5.4) and MBS (OR: 4.1, 95% CI: 1.8–9.2) were significantly associated with atopic dermatitis. In preschool children older than 2 years, both MBD and MBS may be associated with the onset or development of atopic dermatitis.     

Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka)

Most studies assessing adaptive MHC diversity in salmon populations have focused on the classical class II DAB or DAA loci, as these have been most amenable to single PCR amplifications due to their relatively low level of sequence divergence. Herein, we report the characterization of the classical class I UBA α2 locus based on collections taken throughout the species range of sockeye salmon (Oncorhynchus nerka). Through use of multiple lineage-specific primer sets, denaturing gradient gel electrophoresis and sequencing, we identified thirty-four alleles from three highly divergent lineages. Sequence identity between lineages ranged from 30.0% to 56.8% but was relatively high within lineages. Allelic identity within the antigen recognition site (ARS) was greater than for the longer sequence. Global positive selection on UBA was seen at the sequence level (dN:dS = 1.012) with four codons under positive selection and 12 codons under negative selection.     

Serological survey of equine viral diseases in Mongolia

Three hundred sera were collected from horses in various parts of Mongolia in 2007 and seroepidemiological surveys for several equine viruses performed on them. Equid herpesvirus 1 and equine rhinitis A virus were prevalent, and equine arteritis virus and equid herpesvirus 3 were detected over a wide area though their rates of antibody-positivity were not high. Equine infectious anemia was distributed locally. The rates of horses antibody-positive for Japanese encephalitis virus and equine influenza virus were low, but these were detected. Bovine coronavirus antibodies were detected at a high rate, but it was not clear whether they were due to horse coronavirus.     

Autoregulation of nodulation interferes with impacts of nitrogen fertilization levels on the leaf-associated bacterial community in soybeans

The diversities leaf-associated bacteria on nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S rRNA gene. To analyze the impact of nitrogen fertilization on the bacterial leaf community, soybeans were treated with standard nitrogen (SN) (15 kg N ha(-1)) or heavy nitrogen (HN) (615 kg N ha(-1)) fertilization. Under SN fertilization, the relative abundance of Alphaproteobacteria was significantly higher in Nod(-) and Nod(++) soybeans (82% to 96%) than in Nod(+) soybeans (54%). The community structure of leaf-associated bacteria in Nod(+) soybeans was almost unaffected by the levels of nitrogen fertilization. However, differences were visible in Nod(-) and Nod(++) soybeans. HN fertilization drastically decreased the relative abundance of Alphaproteobacteria in Nod(-) and Nod(++) soybeans (46% to 76%) and, conversely, increased those of Gammaproteobacteria and Firmicutes in these mutant soybeans. In the Alphaproteobacteria, cluster analyses identified two operational taxonomic units (OTUs) (Aurantimonas sp. and Methylobacterium sp.) that were especially sensitive to nodulation phenotypes under SN fertilization and to nitrogen fertilization levels. Arbuscular mycorrhizal infection was not observed on the root tissues examined, presumably due to the rotation of paddy and upland fields. These results suggest that a subpopulation of leaf-associated bacteria in wild-type Nod(+) soybeans is controlled in similar ways through the systemic regulation of autoregulation of nodulation, which interferes with the impacts of N levels on the bacterial community of soybean leaves.     

Treatment-Related Death in Patients with Small-Cell Lung Cancer in Phase III Trials over the Last Two Decades

Introduction

Treatment-related death (TRD) remains a serious problem in small-cell lung cancer (SCLC), despite recent improvements in supportive care. However, few studies have formally assessed time trends in the proportion of TRD over the past two decades. The aim of this study was to determine the frequency and pattern of TRD over time.

Methods

We examined phase 3 trials conducted between 1990 and 2010 to address the role of systemic treatment for SCLC. The time trend was assessed using linear regression analysis.

Results

In total, 97 trials including nearly 25,000 enrolled patients were analyzed. The overall TRD proportion was 2.95%. Regarding the time trend, while it was not statistically significant, it tended to decrease, with a 0.138% decrease per year and 2.76% decrease per two decades. The most common cause of death was febrile neutropenia without any significant time trend in its incidence over the years examined (p = 0.139). However, deaths due to febrile neutropenia as well as all causes in patients treated with non-platinum chemotherapy increased significantly (p = 0.033).

Conclusions

The overall TRD rate has been low, but not negligible, in phase III trials for SCLC over the past two decades.