Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings

Background: A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics.

Methods: Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release.

Results: The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency.

Conclusion: These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.     

Genotype-first approach to the detection of hereditary breast and ovarian cancer risk,and effects of risk disclosure to biobank participants

Genotype-first approach allows to systematically identify carriers of pathogenic variants in BRCA1/2 genes conferring a high risk of familial breast and ovarian cancer. Participants of the Estonian biobank have expressed support for the disclosure of clinically significant findings. With an Estonian biobank cohort, we applied a genotype-first approach, contacted carriers, and offered return of results with genetic counseling. We evaluated participants’ responses to and the clinical utility of the reporting of actionable genetic findings. Twenty-two of 40 contacted carriers of 17 pathogenic BRCA1/2 variants responded and chose to receive results. Eight of these 22 participants qualified for high-risk assessment based on National Comprehensive Cancer Network criteria. Twenty of 21 counseled participants appreciated being contacted. Relatives of 10 participants underwent cascade screening. Five of 16 eligible female BRCA1/2 variant carriers chose to undergo risk-reducing surgery, and 10 adhered to surveillance recommendations over the 30-month follow-up period. We recommend the return of results to population-based biobank participants; this approach could be viewed as a model for population-wide genetic testing. The genotype-first approach permits the identification of individuals at high risk who would not be identified by application of an approach based on personal and family histories only.Subject terms: Genetic counselling, Population screening, Cancer genomics     

Increasing participation in cervical cancer screening: Telephone contact with long‐term non‐attendees in Sweden. Results from RACOMIP,a randomized controlled trial

Non‐participation is the foremost screening‐related risk factor for cervical cancer. We studied the effectiveness and cost‐effectiveness of an intervention to increase participation in the context of a well‐run screening program. Telephone contact with non‐attendees, offering an appointment to take a smear, was compared with a control group in a population‐based randomized trial in western Sweden. Of 8,800 randomly selected women aged 30–62, without a registered Pap smear in the two latest screening rounds, 4,000 were randomized to a telephone arm, another 800 were offered a high‐risk human papillomavirus (HPV) self‐test by mail (not reported in this article) and 4,000 constituted a control group. Endpoints were frequency of testing, frequency of abnormal smears and further assessment of abnormal tests. Participation during the following 12 months was significantly higher in the telephone arm than in the control group, 718 (18.0%) versus 422 (10.6%) [RR: 1.70, 95% confidence interval (CI): 1.52–1.90]. The number of detected abnormal smears was 39 and 19, respectively (RR: 2.05, 95% CI: 1.19–3.55). The respective numbers of further assessed abnormalities were 34 and 18 (RR: 1.89, 95% CI: 1.07–3.34). Twice as many high‐grade intraepithelial neoplasia (CIN2+) were detected and treated in the telephone arm: 14 and 7, respectively. Telephone contact with women who have abstained from cervical cancer screening for long time increases participation and leads to a significant increase in detection of atypical smears. Cost calculations indicate that this intervention is unlikely to be cost‐generating and this strategy is feasible in the context of a screening program.     

Quantification of Clostridium difficile in antibiotic-associated-diarrhea patients

Comparing culture- and non-culture-based methods for quantifying Clostridium difficile in antibiotic-associated-diarrhea patients, we found that the real-time PCR method correlated well with quantitative culture and was more sensitive. A positive association between the population levels of C. difficile and the presence of its toxins was found.     

Effects of serotonin transporter promoter and BDNF Val66Met genotype on personality traits in a population representative sample of adolescents

The purpose of this study was to investigate the effects of the 5-HTTLPR and brain-derived neurotrophic factor (BDNF) Val66Met polymorphisms on self-reported Big Five personality traits and their facets in a population representative sample of adolescents. The sample consisted of both cohorts of the Estonian Children Personality Behaviour and Health Study, and personality data were collected during its second waves. The 5-HTTLPR and BDNF Val66Met polymorphisms were genotyped. The BDNF Val66Met had a significant effect on conscientiousness [F(1,807)=4.32, P=0.038]. We did not find effects of the 5-HTTLPR polymorphism on the main domains of personality, however, a gene×gene interaction on conscientiousness emerged -BDNF Val66Met Met-allele carriers with the 5-HTTLPR s/s genotype had by far the lowest scores in conscientiousness [F(2,803)=4.38, P=0.012]. In addition, we found genotype effects on some facet scales. In conclusion, the BDNF Val66Met genotype Met-allele carriers have lower conscientiousness, and this effect is increased in the 5-HTTLPR s/s individuals.     

Functional micro-ultrasound imaging of rodent cerebral hemodynamics

Healthy cerebral microcirculation is crucial to neuronal functioning. We present a new method to investigate microvascular hemodynamics in living rodent brain through a focal cranial window based on high-frequency ultrasound imaging. The method has a temporal resolution of 40ms, and a 100μm in-plane and 600μm through-plane spatial resolution. We use a commercially available high-frequency ultrasound imaging system to quantify changes in the relative cerebral blood volume (CBV) by measuring the scattered signal intensity from an ultrasound contrast agent circulating in the vasculature. Generalized linear model analysis is then used to produce effect size and significance maps of changes in cerebral blood volume upon electrical stimulation of the forepaw. We observe larger CBV increases in the forelimb representation of the primary somatosensory cortex than in the deep gray matter with stimuli as short as 2s (5.1 ± 1.3% vs. 3.3 ± 0.6%). We also investigate the temporal evolution of the blood volume changes in cortical and subcortical gray matter, pial vessels and subcortical major vessels, and show shorter response onset times in the parenchymal regions than in the neighboring large vessels (1.6 ± 1.0s vs. 2.6 ± 1.3s in the cortex for a 10 second stimulus protocol). This method, which we termed functional micro-ultrasound imaging or fMUS, is a novel, highly accessible, and cost-effective way of imaging rodent brain microvascular topology and hemodynamics in vivo at 100micron resolution over a 1-by-1cm field of view with 10s-100s frames per second that opens up a new set of questions regarding brain function in preclinical models of health and disease.     

A constrained independent component analysis technique for artery-vein separation of two-photon laser scanning microscopy images of the cerebral microvasculature

Understanding brain hemodynamics as well as the coupling between microvascular hemodynamics and neural activity is important in pathophysiology of cerebral microvasculature. When local increases in neuronal activity occur, the blood volume changes in the surrounding brain vasculature. Dynamic contrast enhanced imaging (DCE) is a powerful technique that quantifies these changes in the blood flow by repeatedly imaging the vasculature over time. Separating artery, vein and capillaries in the images and extracting their intensity-time curves from the DCE image sequence is an important first step in understanding vascular function. A constrained independent component analysis (ICA) technique is developed to analyze the two photon laser scanning microscopy (2PLSM) images of rat brain microvasculature, where a bolus of fluorescent dye is administered to the vascular system as the contrast agent. A priori information inferred from the gamma variate model of cerebral microvasculature is incorporated with the data driven technique in temporal and spatial domains using two constraints. The constraints are: no independent component (IC) is allowed to have negative contribution in forming the images (positivity constraint) and the component curves follow a gamma variate function (model fitting constraint). Experimental and simulation studies are conducted to demonstrate the improved performance of the proposed constrained ICA (CICA) technique over the most commonly used classical ICA algorithm (fast-ICA) in providing physiologically meaningful ICs and its ability to separate the model following factors from other factors are shown. The efficiency of CICA in handling noise is compared to model based techniques. Its capability in providing improved separation between artery, vein and capillaries compared to the other two techniques is also demonstrated.     

A CASE OF DIFFUSE‐TYPE EARLY GASTRIC CANCER WITH NODULAR GASTRITIS

A 39‐year‐old woman was referred to Osaka Police Hospital and admitted for surgical treatment of gastric cancer. Barium upper gastrointestinal study and endoscopic examination showed a 3.0 × 3.0 cm depressed lesion in the greater curvature of the middle corpus. An unusual miliary pattern resembling ‘goose flesh’ was observed endoscopically in the antrum. Biopsy specimens from the tumor showed poorly differentiated adenocarcinoma, and specimens from the antrum showed many lymphoid follicles with a germinal center. Rapid urease test and histological tests (Giemsa stain) for Helicobacter pylori were both positive. Early gastric cancer with nodular gastritis (NG) was diagnosed and a partial gastrectomy was performed. Histological examination of the resected specimen showed a stage I tumor consisting mainly of signet‐ring cell carcinoma restricted to the mucosa. Postoperatively H. pylori eradication therapy was performed and proved to be successful. One year after eradication therapy, endoscopy with biopsy showed no recurrence of gastric cancer and the remarkable regression of antral NG.     

Low Prevalence of Asthma in Westernizing Countries—Myth or Reality? Prevalence of Asthma in Estonia—A Report from the “FinEsS” Study

Aim: The aim of this study was to examine the prevalence of asthma among adults in Estonia by using different diagnostic methods and criteria for the disease. Methods: In 1995-1996, a postal questionnaire was randomly distributed in three areas of Estonia to a representative sample of 22,579 subjects aged 15-64 years. The participation rate was 78%. A clinical follow-up study including structured interview, skin prick test, lung function, and methacholine test, was performed from 1997 to 2000 among randomly selected responders to the postal questionnaire. Of 2676 subjects, 53% participated. Results: The prevalence of physician-diagnosed asthma for the postal questionnaire (PQ) was 2.7%, and in the same subjects for the structured interview (SI) 3.8%. Respiratory symptoms, except recurrent wheeze, were more common in the SI than PQ. Combinations of symptoms, except wheezing with breathlessness apart from cold, were more prevalent in the PQ responders. The prevalence of asthma defined by different symptom combinations varied from 5.4% to 8.2%. Among responders, 71-87% demonstrated bronchial hyperreactivity defined as methacholine reactivity ≤ 8 mg/mL. The symptom combinations used as surrogate variables for asthma were strongly associated with a positive skin test. Conclusion: The low prevalence of physician-diagnosed asthma probably reflects a considerable underdiagnosis of asthma in Estonia. Disease criteria for asthma based on symptom combinations together with hyperreactivity yielded a prevalence of 5%-8%, which is similar to the prevalence of asthma among adults in neighboring Western countries.