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Identification of genomic variability in population plays an important role in the clinical diagnostics of human genetic diseases. Thanks to rapid technological development in the field of massive parallel sequencing technologies, also known as next-generation sequencing (NGS), complex genomic analyses are now easier and cheaper than ever before, which consequently leads to more effective utilization of these techniques in clinical practice. However, interpretation of data from NGS is still challenging due to several issues caused by natural variability of DNA sequences in human populations. Therefore, development and realization of projects focused on description of genetic variability of local population (often called “national or digital genome”) with a NGS technique is one of the best approaches to address this problem. The next step of the process is to share such data via publicly available databases. Such databases are important for the interpretation of variants with unknown significance or (likely) pathogenic variants in rare diseases or cancer or generally for identification of pathological variants in a patient’s genome. In this paper, we have compiled an overview of published results of local genome sequencing projects from United Kingdom and Europe together with future plans and perspectives for newly announced ones.

•We investigated a herpes zoster vaccine candidate at different antigen doses, and with or without the AS01B Adjuvant System.•All adjuvanted vaccine formulations were immunogenic and well-tolerated in adults ≥60 years of age.•Two vaccinations with the adjuvanted vaccine candidate induced a greater immune response than one.•The dose of varicella-zoster virus glycoprotein E impacted humoral but not cellular immune responses. This study investigated the safety and immunogenicity of different formulations and schedules of a candidate subunit herpes zoster vaccine containing varicella-zoster virus glycoprotein E (gE) with or without the adjuvant system AS01B. In this phase II, single-blind, randomized, controlled study, adults aged ≥60years (N=714) received one dose of 100μggE/AS01B, two doses, two months apart, of 25, 50, or 100μggE/AS01B, or two doses of unadjuvanted 100μggE/saline. Frequencies of CD4+ T cells expressing ≥2 activation markers following induction with gE were measured by intracellular cytokine staining and serum anti-gE antibody concentrations by ELISA. Frequencies of gE-specific CD4+ T cells were >3-fold higher after two doses of all gE/AS01B formulations than after one dose of 100μggE/AS01B or two doses of 100μggE/saline. Frequencies were comparable after two doses of 25, 50, or 100μggE/AS01B. Serum anti-gE antibody concentrations were comparable after two doses of 50 or 100μggE/AS01B and higher than in the other groups. Immune responses persisted for at least 36 months. Reactogenicities of all gE/AS01B formulations were similar but greater than with gE/saline. The three formulations of gE/AS01B were immunogenic and well tolerated in adults aged ≥60years. Two vaccinations with gE/AS01B induced higher immune responses than one and the dose of gE impacted humoral but not cellular immune responses (NCT00434577).

Abstract Background The herpes zoster subunit vaccine (HZ/su), consisting of varicella-zoster virus glycoprotein E (gE) and AS01B Adjuvant System, was highly efficacious in preventing herpes zoster in the ZOE-50 and ZOE-70 trials. We present immunogenicity results from those trials. Methods Participants (ZOE-50: ≥50; ZOE-70: ≥70 years of age) received 2 doses of HZ/su or placebo, 2 months apart. Serum anti-gE antibodies and CD4 T cells expressing ≥2 of 4 activation markers assessed (CD42+) after stimulation with gE-peptides were measured in subcohorts for humoral (n = 3293) and cell-mediated (n = 466) immunogenicity. Results After vaccination, 97.8% of HZ/su and 2.0% of placebo recipients showed a humoral response. Geometric mean anti-gE antibody concentrations increased 39.1-fold and 8.3-fold over baseline in HZ/su recipients at 1 and 36 months post-dose 2, respectively. A gE-specific CD42+ T-cell response was shown in 93.3% of HZ/su and 0% of placebo recipients. Median CD42+ T-cell frequencies increased 24.6-fold (1 month) and 7.9-fold (36 months) over baseline in HZ/su recipients and remained ≥5.6-fold above baseline in all age groups at 36 months. The proportion of CD4 T cells expressing all 4 activation markers increased over time in all age groups. Conclusions Most HZ/su recipients developed robust immune responses persisting for 3 years following vaccination. Clinical Trials Registration NCT01165177; NCT01165229. The herpes zoster subunit vaccine, consisting of varicella-zoster virus glycoprotein E and the AS01B Adjuvant System, stimulated specific antibody and CD4 T-cell responses in >90% of recipients which, in most, persisted for the 36-month duration of the study.

Although fever is part of the normal inflammatory process after immunisation, prophylactic antipyretic drugs are sometimes recommended to allay concerns of high fever and febrile convulsion. We assessed the effect of prophylactic administration of paracetamol at vaccination on infant febrile reaction rates and vaccine responses. In two consecutive (primary and booster) randomised, controlled, open-label vaccination studies, 459 healthy infants were enrolled from ten centres in the Czech Republic. Infants were randomly assigned with a computer-generated randomisation list to receive three prophylactic paracetamol doses every 6–8 h in the first 24 h (n=226) or no prophylactic paracetamol (n=233) after each vaccination with a ten-valent pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) co-administered with the hexavalent diphtheria-tetanus-3-component acellular pertussis-hepatitis B-inactivated poliovirus types 1, 2, and 3- H influenzae type b (DTPa-HBV-IPV/Hib) and oral human rotavirus vaccines. The primary objective in both studies was the reduction in febrile reactions of 38·0°C or greater in the total vaccinated cohort. The second objective was assessment of immunogenicity in the according-to-protocol cohort. These studies are registered with ClinicalTrials.gov, numbers NCT00370318 and NCT00496015. Fever greater than 39·5°C was uncommon in both groups (after primary: one of 226 participants [<1%] in prophylactic paracetamol group vs three of 233 [1%] in no prophylactic paracetamol group; after booster: three of 178 [2%] vs two of 172 [1%]). The percentage of children with temperature of 38°C or greater after at least one dose was significantly lower in the prophylactic paracetamol group (94/226 [42%] after primary vaccination and 64/178 [36%] after booster vaccination) than in the no prophylactic paracetamol group (154/233 [66%] after primary vaccination and 100/172 [58%] after booster vaccination). Antibody geometric mean concentrations (GMCs) were significantly lower in the prophylactic paracetamol group than in the no prophylactic paracetamol group after primary vaccination for all ten pneumococcal vaccine serotypes, protein D, antipolyribosyl-ribitol phosphate, antidiphtheria, antitetanus, and antipertactin. After boosting, lower antibody GMCs persisted in the prophylactic paracetamol group for antitetanus, protein D, and all pneumococcal serotypes apart from 19F. Although febrile reactions significantly decreased, prophylactic administration of antipyretic drugs at the time of vaccination should not be routinely recommended since antibody responses to several vaccine antigens were reduced. GlaxoSmithKline Biologicals (Belgium).

In recent years, Europe has recorded an increase in the number of measles outbreaks despite the implementation of vaccination into the National Immunization Programs. The Czech Republic introduced vaccination against measles into National Immunization Program in 1969. The aim of this study was to determine seroprevalence of IgG antibodies against measles in adults. Our study was designed as a prospective, multicenter cohort study. Samples of blood were taken from adults aged 18 years and over. Specific IgG antibodies were determined by ELISA method. A number of 1911 sera samples were obtained. The total seropositivity reached 83.3%, 14.3% of the results were negative and 2.4% were borderline. When comparing the individual age groups, the highest antibody seropositivity (> 96%) was detected in persons aged 50 years and over who were naturally infected in pre-vaccine era. The lowest seropositivity was recorded in the age groups 30-39 years (61.5%), 40-49 years (77.5%) and 18-29 years (81.1%). A long term high rate of seropositivity persists after natural measles infection. By contrast, it decreases over time after vaccination. Similarly, the concentrations of antibodies in persons with measles history persist for a longer time at a higher level than in vaccinated persons. Our results indicate possible gap in measles protection in adults born after implementation of vaccination into the National Immunization Programs. There are two probable reasons, decrease of measles antibody seropositivity in time after vaccination in setting of limited natural booster and one-dose vaccination schedule used in the first years after implementation.

Introduction: Intrauterine insemination is a basic method of assisted reproduction. It enables direct deposition of sperm inside the uterine cavity. The complications are rare. Multiple pregnancy or ovarian hyperstimulation can occur when concomitant ovulation induction is performed. The risk of pelvic inflammatory disease due to the artificial cervical barrier breach is very low, but the possible consequences are serious. Case presentation: We present a case of recurrent pelvic inflammatory disease following consecutive intrauterine inseminations. Pelvic inflammatory disease pathophysiology and treatment approaches are discussed. Conclusion: Inflammatory complications of the IUI are rare, but the possible impact on fertility can be devastating. Prevention of the PID together with prompt diagnosis and individualized therapy should be assured to preserve fertility.

Background Neurodevelopmental disorders (NDDs) and/or associated multiple congenital abnormalities (MCAs) represent a genetically heterogeneous group of conditions with an adverse prognosis for the quality of intellectual and social abilities and common daily functioning. The rapid development of exome sequencing (ES) techniques, together with trio-based analysis, nowadays leads to up to 50% diagnostic yield. Therefore, it is considered as the state-of-the-art approach in these diagnoses. Results In our study, we present the results of ES in a cohort of 85 families with 90 children with severe NDDs and MCAs. The interconnection of the in-house bioinformatic pipeline and a unique algorithm for variant prioritization resulted in a diagnostic yield of up to 48.9% (44/90), including rare and novel causative variants (41/90) and intragenic copy-number variations (CNVs) (3/90). Of the total number of 47 causative variants, 53.2% (25/47) were novel, highlighting the clinical benefit of ES for unexplained NDDs. Moreover, trio-based ES was verified as a reliable tool for the detection of rare CNVs, ranging from intragenic exon deletions ( GRIN2A, ZC4H2 genes) to a 6-Mb duplication. The functional analysis using PANTHER Gene Ontology confirmed the involvement of genes with causative variants in a wide spectrum of developmental processes and molecular pathways, which form essential structural and functional components of the central nervous system. Conclusion Taken together, we present one of the first ES studies of this scale from the central European region. Based on the high diagnostic yield for paediatric NDDs in this study, 48.9%, we confirm trio-based ES as an effective and reliable first-tier diagnostic test in the genetic evaluation of children with NDDs.

Chronic hepatitis C is curable disease. Low detection rate could be one of the reasons of poor treatment uptake. It is important to identify HCV prevalence and anti-hepatitis C virus (HCV) positive patients in population by effective screening strategy such as risk-based or birth cohort screening programs. There are no national population-based estimates of the HCV prevalence in the Czech Republic (CZ). The most recent seroprevalence survey determined a prevalence of positive anti-HCV antibodies of 0.2% (in 2001). The aim of the study was to determine the seroprevalence of HCV, HCV viraemia and HCV genotype in the CZ adult population. We also estimated the number of persons living with chronic hepatitis C in CZ. The examined group included 3000 adults, 18-90 years of age enrolled in 2015. All serum samples were examined to determined anti-HCV antibodies positivity, HCV-RNA positivity and genotypes. Of the 3000 samples, 50 were found to be anti-HCV-positive, for a seroprevalence of 1.67% (2.39% in males, 0.98% in females). The overall prevalence of positive HCV RNA was 0.93%: 1.5% in males, 0.39% in females. HCV genotype (GT) 1a was determined in 25%, GT 1b in 25% and GT 3a in 46%. Since 2001, the HCV seroprevalence has increased 8-fold. The highest HCV seroprevalence occurred in males aged 30-44 years. We can estimate that there are more than 140,000 people with HCV antibodies and more than 80,000 people with chronic hepatitis C living in the CZ. The introduction of birth cohort HCV screening could be beneficial for the country.

Pathogenic sequence variant in the GNAI1 gene were recently introduced as a cause of novel syndrome with a manifestation of variable developmental delay and autistic features. In our study, we report a case of monozygotic twins with severe intellectual disability and motor delay and developmental dysphasia. Both probands and their parents were examined using multi-step molecular diagnostic algorithm including whole-exome sequencing (WES), resulting in the identification of a novel, de novo pathogenic sequence variant in the GNAI1 gene, NM_002069.6:c.815 A>G, p.(Asp272Gly) in probands. Using WES we also verified the microarray findings of a familial 8q24.23q24.3 duplication and heterozygous 5q13.2 deletion, not associated with clinical symptoms in probands. Our results confirmed the role of the GNAI1 gene in the pathogenesis of syndromic neurodevelopmental disorders. They support trio- or quatro-based WES as a suitable molecular diagnostics method for the simultaneous detection of clinically relevant sequence variants and CNVs in individuals with neurodevelopmental disorders and rare diseases.

Background Chromosomal microarray analysis has been shown to be a valuable and cost effective assay for elucidating copy number variants (CNVs) in children with intellectual disability and developmental delay (ID/DD). Methods In our study, we performed array-based comparative genomic hybridization (array-CGH) analysis using oligonucleotide-based platforms in 542 Czech patients with ID/DD, autism spectrum disorders and multiple congenital abnormalities. Prior to the array-CGH analysis, all the patients were first examined karyotypically using G-banding. The presence of CNVs and their putative derivation was confirmed using fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and predominantly relative quantitative polymerase chain reaction (qPCR). Results In total, 5.9% (32/542) patients were positive for karyotypic abnormalities. Pathogenic/likely pathogenic CNVs were identified in 17.7% of them (96/542), variants of uncertain significance (VOUS) were detected in 4.8% (26/542) and likely benign CNVs in 9.2% of cases (50/542). We identified 6.6% (36/542) patients with known recurrent microdeletion (24 cases) and microduplication (12 cases) syndromes, as well as 4.8% (26/542) patients with non-recurrent rare microdeletions (21 cases) and microduplications (5 cases). In the group of patients with submicroscopic pathogenic/likely pathogenic CNVs (13.3%; 68/510) we identified 91.2% (62/68) patients with one CNV, 5.9% (4/68) patients with two likely independent CNVs and 2.9% (2/68) patients with two CNVs resulting from cryptic unbalanced translocations. Of all detected CNVs, 21% (31/147) had a de novo origin, 51% (75/147) were inherited and 28% (41/147) of unknown origin. In our cohort pathogenic/likely pathogenic microdeletions were more frequent than microduplications (69%; 51/74 vs. 31%; 23/74) ranging in size from 0.395 Mb to 10.676 Mb (microdeletions) and 0.544 Mb to 8.156 Mb (microduplications), but their sizes were not significantly different ( P  = 0.83). The pathogenic/likely pathogenic CNVs (median 2.663 Mb) were significantly larger than benign CNVs (median 0.394 Mb) ( P  < 0.00001) and likewise the pathogenic/likely pathogenic CNVs (median 2.663 Mb) were significantly larger in size than VOUS (median 0.469 Mb) ( P  < 0.00001). Conclusions Our results confirm the benefit of array-CGH in the current clinical genetic diagnostics leading to identification of the genetic cause of ID/DD in affected children.