Evaluation of auditory pathway excitability using a pre-operative trans-tympanic electrically evoked auditory brainstem response under local anesthesia in cochlear implant candidates.

OBJECTIVE: Subjective promontory stimulation is used to evaluate cochlear implant (CI) candidacy, but the test reliability is low. Electrically evoked auditory brainstem response (EABR) can verify the function of the auditory system objectively. This study's procedure uses a trans-tympanic rounded bent-tip electrode to perform pre-operative EABR under local anaesthesia (LA-TT-EABR) using MED-EL Software and Hardware. This study aimed to determine usability and effectiveness for CI candidates. DESIGN: We hypothesised that LA-TT-EABR waveforms of good quality would be related to successful hearing outcomes. We assumed that the duration of hearing loss/deafness was a confounding factor to study outcomes. STUDY SAMPLE: 19 borderline CI candidates. RESULTS: Positive LA-TT-EABR results were confirmed in 14 patients. LA-TT-EABR's mean latency was 2.05 ± 0.31 ms (eII/eIII) and 4.24 ± 0.39 ms (eIV/eV). Latencies weren't statistically different from intra-operative EABR elicited by basal CI contacts. All positive LA-TT-EABR patients benefitted from CI and speech performance improved one year after implantation. One patient with negative LA-TT-EABR was cochlear-implanted and had no hearing sensation. CONCLUSIONS: LA-TT-EABR is a tool in the frame of pre-operative objective testing the auditory pathway. It seems useful for clinical testing CI candidacy. Based on this study's outcomes, LA-TT-EABR should be recommended for uncertain CI candidates.

Carrier dynamics in (Ga,In)(Sb,Bi)/GaSb quantum wells for laser applications in the mid-infrared spectral range.

We present experimental studies on low-temperature ([Formula: see text]) carrier dynamics in (Ga,In)(Sb,Bi)/GaSb quantum wells (QWs) with the nominal In content of 3.7% and the Bi ranging from 6 to 8%. The photoreflectance experiment revealed the QW bandgap evolution with [Formula: see text] % Bi, which resulted in the bandgap tunability roughly between 629 and [Formula: see text], setting up the photon emission wavelength between 1.97 and [Formula: see text]. The photoluminescence experiment showed a relatively small 3-10[Formula: see text] Stokes shift regarding the fundamental QW absorption edge, indicating the exciton localisation beneath the QW mobility edge. The localised state's distribution, being the origin of the PL, determined carrier dynamics in the QWs probed directly by the time-resolved photoluminescence and transient reflectivity. The intraband carrier relaxation time to the QW ground state, following the non-resonant excitation, occurred within 3-25[Formula: see text] and was nearly independent of the Bi content. However, the interband relaxation showed a strong time dispersion across the PL emission band and ranging nearly between 150 and [Formula: see text], indicating the carrier transfer among the localised state's distribution. Furthermore, the estimated linear dispersion variation parameter significantly decreased from [Formula: see text] to [Formula: see text] with increasing the Bi content, manifested the increasing role of the non-radiative recombination processes with Bi in the QWs.

Neonatal screening for congenital hypothyroidism: Time to lower the TSH threshold in France.

Neonatal screening for congenital hypothyroidism (CH) is based on the measurement of thyroid-stimulating hormone (TSH) in whole dried blood samples on filter paper in all newborns. The objective of screening for CH is to prevent mental retardation, which is irreversible in the event of a late diagnosis, by setting up prompt treatment (before day 15) with levothyroxine. The threshold value of TSH on filter paper on day 3 is 17 mIU/L in France in the GSP method (GSP, Genetic Screening Processor, Perkin Elmer): It is one of the highest thresholds used in the world. In many countries, the TSH threshold is between 6 and 12 mIU/L. Studies have found that a threshold of > 17 mIU/L may miss as much as 30% of cases of CH, with 30-80% of these being permanent CH. Recent studies suggest that mild CH (currently missed by the French TSH threshold) is associated with cognitive consequences if left untreated. An inverse relationship between TSH at screening (below the current threshold) and cognitive development at preschool or school age has been shown. These studies advocate for the evaluation of a lowering of the threshold of TSH on filter paper in France: (a) to determine the number of CH diagnoses with the new threshold and whether these "new cases" would be transitory or permanent; and (b) to analyze the cost-effectiveness of the strategy.

Adherence and quality of life in children receiving rhGH treatment.

Short stature may have a major impact on quality of life (QoL), not only during adulthood but also during childhood. Treatment by growth hormone may induce improvement in QoL through height gain, as shown in recent articles, with an increase in general health-related and also height-specific QoL assessed by self-reports and parental reports. In a paper published by our team, we show altered general-health QoL in patients with very short stature (≤ -3 SD) and an improvement in general and height-specific scales in the complete population (≤ -2 SD) after one year of recombinant human growth hormone (rhGH) treatment, perceived both by children and their parents, with a moderate positive correlation with height gain. Adequate results in terms of height gain depend on different factors: the patient's age, underlying condition for which rhGH is prescribed and dose of rhGH treatment, among others. Daily injections may cause a significant burden for the child and family, and may alter adequate adherence to treatment. Identifying positive and negative factors in the patient and in the healthcare providers-patient team and encouraging a shared decision-making process are important for improving the patient's adherence to treatment. New long-acting forms of rhGH that will be available in the next few years may play an important part in improving treatment-related QoL and adherence to treatment. © 2022 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Machine learning applied to atopic dermatitis transcriptome reveals distinct therapy-dependent modification of the keratinocyte immunophenotype.

BACKGROUND: Atopic dermatitis (AD) arises from a complex interaction between an impaired epidermal barrier, environmental exposures, and the infiltration of T helper (Th)1/Th2/Th17/Th22 T cells. Transcriptomic analysis has advanced our understanding of gene expression in cells and tissues. However, molecular quantitation of cytokine transcripts does not predict the importance of a specific pathway in AD or cellular responses to different inflammatory stimuli. OBJECTIVES: To understand changes in keratinocyte transcriptomic programmes in human cutaneous disease during development of inflammation and in response to treatment. METHODS: We performed in silico deconvolution of the whole-skin transcriptome. Using co-expression clustering and machine-learning tools, we resolved the gene expression of bulk skin (seven datasets, n = 406 samples), firstly, into keratinocyte phenotypes identified by unsupervised clustering and, secondly, into 19 cutaneous cell signatures of purified populations from publicly available datasets. RESULTS: We identify three unique transcriptomic programmes in keratinocytes - KC1, KC2 and KC17 - characteristic of immune signalling from disease-associated Th cells. We cross-validate those signatures across different skin inflammatory conditions and disease stages and demonstrate that the keratinocyte response during treatment is therapy dependent. Broad-spectrum treatment with ciclosporin ameliorated the KC17 response in AD lesions to a nonlesional immunophenotype, without altering KC2. Conversely, the specific anti-Th2 therapy, dupilumab, reversed the KC2 immunophenotype. CONCLUSIONS: Our analysis of transcriptomic signatures in cutaneous disease biopsies reveals the effect of keratinocyte programming in skin inflammation and suggests that the perturbation of a single axis of immune signal alone may be insufficient to resolve keratinocyte immunophenotype abnormalities.

Diagnostic performance of axillary ultrasound and standard breast MRI for differentiation between limited and advanced axillary nodal disease in clinically node-positive breast cancer patients.

Preoperative differentiation between limited (pN1; 1-3 axillary metastases) and advanced (pN2-3; ≥4 axillary metastases) nodal disease can provide relevant information regarding surgical planning and guiding adjuvant radiation therapy. The aim was to evaluate the diagnostic performance of preoperative axillary ultrasound (US) and breast MRI for differentiation between pN1 and pN2-3 in clinically node-positive breast cancer. A total of 49 patients were included with axillary metastasis confirmed by US-guided tissue sampling. All had undergone breast MRI between 2008-2014 and subsequent axillary lymph node dissection. Unenhanced T2-weighted MRI exams were reviewed by two radiologists independently. Each lymph node on the MRI exams was scored using a confidence scale (0-4) and compared with histopathology. Diagnostic performance parameters were calculated for differentiation between pN1 and pN2-3. Interobserver agreement was determined using Cohen's kappa coefficient. At final histopathology, 67.3% (33/49) and 32.7% (16/49) of patients were pN1 and pN2-3, respectively. Breast MRI was comparable to US in terms of accuracy (MRI reader 1 vs US, 71.4% vs 69.4%, p = 0.99; MRI reader 2 vs US, 73.5% vs 69.4%, p = 0.77). In the case of 1-3 suspicious lymph nodes, pN2-3 was observed in 30.4% on US (positive predictive value (PPV) 69.6%) and in 22.2-24.3% on MRI (PPV 75.7-77.8%). In the case of ≥4 suspicious lymph nodes, pN1 was observed in 33.3% on US (negative predictive value (NPV) 66.7%) and in 38.5-41.7% on MRI (NPV 58.3-61.5%). Interobserver agreement was considered good (k = 0.73). In clinically node-positive patients, the diagnostic performance of axillary US and breast MRI is comparable and limited for accurate differentiation between pN1 and pN2-3. Therefore, there seems no added clinical value of preoperative breast MRI regarding nodal staging in patients with positive axillary US.

Novel method of fitting of children with auditory brainstem implants.

OBJECTIVES: To develop a reliable and objective fitting method for use with young children with an auditory brainstem implant (ABI). MATERIALS AND METHODS: Subjects were 17 young children implanted with an ABI with the mean age 2 years and 4 months (8-64 months). Evoked auditory brainstem response (eABR) measurements were performed intraoperatively and at activation in order to record the auditory response and non-auditory side effects. Each child was tested to observe any subjective responses to the electric stimuli and non-auditory side effects. All children were fitted based on the postoperative eABR. The minimum follow up time was 12 months. RESULTS: Intraoperatively an eABR could be obtained in all children. The responses were recordable from 75-100% of all electrodes. At initial stimulation eABR were recordable in all children. The eABR was obtained in 79.7% of all electrodes (25-100%) with a mean eABR threshold of 22.3 nC. eABR without any non-auditory stimulation was recorded on all electrodes in 11 children. Mixed eABR and non-auditory responses were recorded on 2-6 electrodes in 6 children. The subjective auditory responses for at least 1 electrode were noted in 15 children. In the 2 remaining cases the auditory response was obtained only when the device was activated. In all children the subjective responses were within the estimated dynamic range for each electrode. Each child was able to accept up to 100% of volume of the created map. The non-auditory response was observed only on children and electrodes with mixed eABR and non-auditory responses. The mean CAP score at 6 months after the activation was 2.4 (1-4). CONCLUSIONS: eABR seems to be a reliable tool to judge ABI electrode placement and a reliable method for fitting of young children with an ABI. The data suggest that eABR-based fitting helps children to more quickly achieve auditory perception and development.

Impact of transitional care on endocrine and anthropometric parameters in Prader-Willi syndrome.

CONTEXT: The transition of patients with Prader-Willi syndrome (PWS) to adult life for medical care is challenging because of multiple comorbidities, including hormone deficiencies, obesity and cognitive and behavioral disabilities. OBJECTIVE: To assess endocrine management, and metabolic and anthropometric parameters of PWS adults who received (n = 31) or not (n = 64) transitional care, defined as specialized pediatric care followed by a structured care pathway to a multidisciplinary adult team. PATIENTS AND STUDY DESIGN: Hormonal and metabolic parameters were retrospectively recorded in 95 adults with PWS (mean ± s.d. age 24.7 ± 8.2 years, BMI: 39.8 ± 12.1 kg/m²) referred to our Reference Center and compared according to transition. RESULTS: Among the entire cohort, 35.8% received growth hormone (GH) during childhood and 16.8% had a GH stimulation test after completion of growth. In adulthood, 14.7% were treated with GH, 56.8% received sex-hormone therapy, whereas 91.1% were hypogonadic and 37.9% had undergone valid screening of the corticotropic axis. The main reason for suboptimal endocrine management was marked behavioral disorders. Patients receiving transitional care were more likely to have had a GH stimulation test and hormonal substitutions in childhood. They also had a lower BMI, percentage of fat mass, improved metabolic parameters and fewer antidepressant treatments. Transitional care remained significantly associated with these parameters in multivariate analysis when adjusted on GH treatment. CONCLUSION: A coordinated care pathway with specialized pediatric care and transition to a multidisciplinary adult team accustomed to managing complex disability including psychiatric troubles are associated with a better health status in adults with PWS.

Choosing between Higher and Lower Resolution Microarrays: do Pregnant Women Have Sufficient Knowledge to Make Informed Choices Consistent with their Attitude?

Developments in prenatal testing allow the detection of more findings. SNP arrays in prenatal diagnosis (PND) can be analyzed at 0.5 Mb resolution detecting more clinically relevant anomalies, or at 5 Mb resolution. We investigated whether women had sufficient knowledge to make informed choices regarding the scope of their prenatal test that were consistent with their attitude. Pregnant women could choose between testing at 5 or at 0.5 Mb array. Consenting women (N = 69) received pre-test genetic counseling by phone and filled out the Measure of Informed Choice questionnaire designed for this study. Choices based on sufficient knowledge and consistent with attitude were considered informed. Sixty-two percent of the women made an adequately informed choice, based on sufficient knowledge and attitude-consistent with their choice of microarray resolution. Women who made an informed choice, opted for 0.5 Mb array resolution more often. There were no differences between women making adequately informed or less informed choices regarding level of experienced anxiety or doubts. Over time on T0 and T1, anxiety and doubts significantly decreased. While previous studies demonstrated that knowledge is an important component in informed decision-making, this study underlines that a consistent attitude might be equally important for decision-making. We advocate more focus on attitude-consistency and deliberation as compared to only a strong focus on knowledge.

Frequency of submicroscopic chromosomal aberrations in pregnancies without increased risk for structural chromosomal aberrations: systematic review and meta-analysis.

OBJECTIVE: To establish, based on a systematic literature review, the frequency of pathogenic submicroscopic chromosomal aberrations in fetuses that are not at increased risk for unbalanced structural chromosomal aberrations, with the aim of determining whether high-resolution testing for submicroscopic aberrations is beneficial in a general pregnant population. METHODS: EMBASE, PubMed, Web of Science and CENTRAL databases were searched systematically on 3 June 2016 for all relevant articles on the prevalence of pathogenic submicroscopic copy number variants (CNVs) in fetuses referred for prenatal invasive testing because of advanced maternal age (AMA) or parental anxiety (ANX). Relevant full-text articles were analyzed and the prevalence of submicroscopic CNVs was calculated based on the extracted data. Meta-analysis was conducted in a pooled cohort of 10 614 fetuses based on the 10 largest studies (n > 300) of a total of 19 that were relevant. RESULTS: Pooled estimate analysis indicated that 0.84% (95% CI, 0.55-1.30%) of fetuses that had invasive testing because of AMA/ANX carried a pathogenic clinically significant submicroscopic aberration. The onset/penetrance of submicroscopic findings was studied in 10 314 fetuses reported in eight papers that presented aberrant cases with all necessary details to allow assessment of the findings. The pooled estimates resulting from meta-analysis of the data indicated that an early-onset syndromic disorder was detected in 0.37% (95% CI, 0.27-0.52%) of cases, a susceptibility CNV was found in 0.30% (95% CI, 0.14-0.67%) and late-onset diseases were reported in 0.11% (95% CI, 0.05%-0.21%). The prevalence of early-onset syndromic disorders caused by a submicroscopic aberration was calculated to be 1:270. When the risk for submicroscopic aberrations is added to the individual risk for microscopic chromosomal aberrations, all pregnant women have a risk of higher than 1 in 180 for a relevant chromosomal aberration, and pregnant women under 36 years of age have a higher risk for submicroscopic pathogenic aberrations than for Down syndrome. CONCLUSION: This systematic review shows that a significant proportion of fetuses in a general pregnant population carry a submicroscopic pathogenic CNV. Based on these figures, all women should be informed on their individual risk for all pathogenic chromosomal aberrations and not only for common trisomies. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Nitrogen-related intermediate band in P-rich GaNxPyAs1-x-y alloys.

The electronic band structure of phosphorus-rich GaNxPyAs1-x-y alloys (x ~ 0.025 and y ≥ 0.6) is studied experimentally using optical absorption, photomodulated transmission, contactless electroreflectance, and photoluminescence. It is shown that incorporation of a few percent of N atoms has a drastic effect on the electronic structure of the alloys. The change of the electronic band structure is very well described by the band anticrossing (BAC) model in which localized nitrogen states interact with the extended states of the conduction band of GaAsP host. The BAC interaction results in the formation of a narrow intermediate band (E- band in BAC model) with the minimum at the Γ point of the Brillouin zone resulting in a change of the nature of the fundamental band gap from indirect to direct. The splitting of the conduction band by the BAC interaction is further confirmed by a direct observation of the optical transitions to the E+ band using contactless electroreflectance spectroscopy.

Chemotherapy may eradicate ductal carcinoma in situ (DCIS) but not the associated microcalcifications.

INTRODUCTION: We studied the effect of neoadjuvant chemotherapy (NAC) ± trastuzumab on the ductal carcinoma in situ (DCIS) component in patients with locally advanced breast cancer who achieved pathological complete response (pCR). METHODS: The diagnostic biopsies of 92 consecutive breast cancer patients that were treated with neoadjuvant chemotherapy (NAC) ± trastuzumab were evaluated for the presence of DCIS. Upon completion of NAC, the surgical specimens were evaluated for complete eradication of both the invasive and non-invasive cancer in the breast. The pretreatment mammograms were evaluated for the presence of microcalcifications and compared to the mammograms that were obtained upon completion of therapy prior to surgery. RESULTS: Thirty of 92 patients (33%) had a substantial component of DCIS in the pretreatment biopsy. Thirty nine patients (42%) achieved pCR: 22 (56%) following NAC + trastuzumab, 17 (32%) following chemotherapy only. Ten of 30 patients (33%) with DCIS component achieved pCR: 4 received chemotherapy only, in 6 trastuzumab was added. Multiple microcalcifications on the pretreatment mammograms were observed in 3 of 10 patients with DCIS who achieved pCR. No reduction in the area of calcifications was observed following NAC. CONCLUSIONS: DCIS may be completely eradicated by NAC ± trastuzumab. However, associated microcalcifications probably persist. Patients with locally advanced breast cancer with substantial DCIS may still opt for NAC and breast conservation as the DCIS component may respond and even completely disappear following NAC. Residual widespread microcalcifications after NAC do not necessarily indicate residual cancer. Larger studies are needed to direct the surgical management of these patients.

Transient neonatal diabetes mellitus and activating mutation in the KCNJ11 gene in two siblings.

Transient neonatal diabetes mellitus is a rare disease usually associated with chromosome 6 abnormalities. Mutations of the genes encoding the potassium channel are rarely associated with these transitional forms. Herein, we report the clinical features of two siblings with a heterozygous mutation C679 G>A in the KCNJ11 gene.

Degradation of PCBs in dry fermented sausages during drying/ripening.

The effects of several commercial meat starter cultures on degradation of polychlorinated biphenyls (PCBs) in dry fermented sausages over 28days of drying/ripening were investigated. The sausage batter was prepared according to a classic recipe and spiked with a standard solution of a PCB congener mixture. With addition of different commercial meat starter cultures, five experimental groups were prepared: no further addition; and separate addition of each of four starter cultures: Texel DCM-1, Texel LM-30, Biostar Sprint, and SM-181. Samples were taken at the beginning of fermentation (zero time), and after 4, 7, 14, 21 and 28days. PCB residues were extracted with hexane. The PCB contents were determined using gas chromatography-mass spectrometry. The PCB levels were reduced in all of the experimental groups tested, where addition of starter culture Biostar Sprint (Lactobacillus sakei, Staphylococcus carnosus, Staphylococcus xylosus) showed the highest PCB degradation rates.

Nano-size scaling of alloy intra-particle vs. inter-particle separation transitions: prediction of distinctly interface-affected critical behaviour.

Phase-separation second-order transitions in binary alloy particles consisting of ∼1000 up to ∼70 000 atoms (∼1-10 nm) are modeled focusing on the unexplored issue of finite-size scaling in such systems, particularly on evaluation of correlation-length critical exponents. Our statistical-thermodynamic approach is based on mean-field analytical expression for the Ising model free energy that facilitates highly efficient computations furnishing comprehensive data for fcc rectangular nanoparticles (NPs). These are summed up in intra- and inter-particle scaling plots as well as in nanophase separation diagrams. Temperature-induced variations in the interface thickness in Janus-type intra-particle configurations and NP size-dependent shifts in the critical temperature of their transition to solid-solution reflect power-law behavior with the same critical exponent, ν = 0.83. It is attributed to dominant interfacial effects that are absent in inter-particle transitions. Variations in ν with nano-size, as revealed by a refined analysis, are linearly extrapolated in order to bridge the gap to larger particles within and well beyond the nanoscale, ultimately yielding ν = 1.0. Besides these findings, the study indicates the key role of the surface-area to volume ratio as an effective linear size, revealing a universal, particle-shape independent, nanoscaling of the critical-temperature shifts.

Pressure coefficients for direct optical transitions in MoS2, MoSe2, WS2, and WSe2 crystals and semiconductor to metal transitions.

The electronic band structure of MoS2, MoSe2, WS2, and WSe2, crystals has been studied at various hydrostatic pressures experimentally by photoreflectance (PR) spectroscopy and theoretically within the density functional theory (DFT). In the PR spectra direct optical transitions (A and B) have been clearly observed and pressure coefficients have been determined for these transitions to be: αA = 2.0 ± 0.1 and αB = 3.6 ± 0.1 meV/kbar for MoS2, αA = 2.3 ± 0.1 and αB = 4.0 ± 0.1 meV/kbar for MoSe2, αA = 2.6 ± 0.1 and αB = 4.1 ± 0.1 meV/kbar for WS2, αA = 3.4 ± 0.1 and αB = 5.0 ± 0.5 meV/kbar for WSe2. It has been found that these coefficients are in an excellent agreement with theoretical predictions. In addition, a comparative study of different computational DFT approaches has been performed and analyzed. For indirect gap the pressure coefficient have been determined theoretically to be -7.9, -5.51, -6.11, and -3.79, meV/kbar for MoS2, MoSe2, WS2, and WSe2, respectively. The negative values of this coefficients imply a narrowing of the fundamental band gap with the increase in hydrostatic pressure and a semiconductor to metal transition for MoS2, MoSe2, WS2, and WSe2, crystals at around 140, 180, 190, and 240 kbar, respectively.

Coffee consumption and risk of hypertension in the Polish arm of the HAPIEE cohort study.

BACKGROUND/OBJECTIVES: Coffee consumption has been hypothesized to be associated with blood pressure (BP), but previous findings are not homogeneous. The aim of this study was to evaluate the association between coffee consumption and the risk of developing hypertension. SUBJECTS/METHODS: Data on coffee consumption, BP and use of anti-hypertensive medicament were derived from 2725 participants of the Polish arm of the HAPIEE project (Health, Alcohol and Psychosocial factors In Eastern Europe) who were free of hypertension at baseline and followed up for an average of 5 years. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by multivariate logistic regression analyses and stratified for potential confounding factors. RESULTS: Coffee consumption was related to decreased age, smoking status and total energy intake. Compared with persons who drink <1 cup coffee per day, systolic BP was significantly associated with coffee consumption and the risk of hypertension was lower for individuals consuming 3-4 cups per day. Despite the analysis stratified by gender showed that the protective effect of coffee consumption on hypertension was significant only in women, the analysis after stratification by smoking status revealed a decreased risk of hypertension in non-smokers drinking 3-4 cups of coffee per day in both sexes (OR 0.41, 95% CI: 0.21, 0.79 for men and OR 0.54, 95% CI: 0.29, 0.99 for women). Upper category coffee consumption (>4 cups per day) was not related to significant increased risk of hypertension. CONCLUSIONS: Relation between coffee consumption and incidence of hypertension was related to smoking status. Consumption of 3-4 cups of coffee per day decreased the risk of hypertension in non-smoking men and women only.

Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2.

AIMS: The aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2. METHODS AND RESULTS: Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5'UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR. CONCLUSIONS: CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report on the application of the CPA method for the detection of BVDV.

Sequence variation in virulence-related genes of Bordetella pertussis isolates from Poland in the period 1959-2013.

This study aimed to characterise Bordetella pertussis isolates circulating in Poland since 1959. Sequence analysis of ptxA, ptxC, prn, tcfA, fim2, fim3 and ptxP for 175 clinical isolates and currently and previously used vaccine strains was performed. Clinical isolates from the period 1995-2013 were found to be different to three currently used vaccine strains harbouring the allelic combination ptxA2-ptxC1-ptxP1-prn1-tcfA2-fim2-1-fim3-1, seen frequently in Poland in the early pertussis vaccination period but not found after 1995. Generally, among B. pertussis isolates from the period 2000-2013, two genotypes predominated, ptxA1-ptxC1-ptxP1-prn1-tcfA2-fim2-2-fim3-1 and ptxA1-ptxC1-ptxP1-prn2-tcfA2-fim2-1-fim3-1, with frequencies of 45% and 32.5%, respectively. The isolates harbouring ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-2 and ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 profiles, currently highly prevalent within other European Union (EU) countries, were rarely found in Poland, as they circulated in the period 2000-2013 with frequencies of 10% and 5%, respectively. We hypothesise that several previous changes of strain composition in whole-cell pertussis vaccine produced locally and used since 1960 in Poland resulted in a more diverse immune pressure in the population, resulting in different prevalence of alleles compared to elsewhere.