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Methylation of host-cell deoxyribonucleic acid (DNA) has been proposed as a promising biomarker for triage of high-risk (hr) human papillomavirus (HPV) positive women at screening. Our study aims to validate recently identified host-cell DNA methylation markers for triage in an hrHPV-positive cohort derived from primary HPV-based cervical screening in The Netherlands. Methylation markers ASCL1, LHX8, ST6GALNAC5, GHSR, ZIC1 and SST were evaluated relative to the ACTB reference gene by multiplex quantitative methylation-specific PCR (qMSP) in clinician-collected cervical samples (n = 715) from hrHPV-positive women (age 29-60 years), who were enrolled in the Dutch IMPROVE screening trial (NTR5078). Primary clinical end-point was cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3+). The single-marker and bi-marker methylation classifiers developed for CIN3 detection in a previous series of hrHPV-positive clinician-collected cervical samples were applied. The diagnostic accuracy was visualised using receiver operating characteristic (ROC) curves and assessed through area under the ROC curve (AUC). The performance of the methylation markers to detect CIN3+ was determined using the predefined threshold calibrated at 70% clinical specificity. Individual methylation makers showed good performance for CIN3+ detection, with highest AUC for ASCL1 (0.844) and LHX8 (0.830). Combined as a bi-marker panel with predefined threshold, ASCL1/LHX8 yielded a CIN3+ sensitivity of 76.9% (70/91; 95% CI 68.3-85.6%) at a specificity of 74.5% (465/624; 95% CI 71.1-77.9%). In conclusion, our study shows that the individual host-cell DNA methylation classifiers and the bi-marker panel ASCL1/LHX8 have clinical utility for the detection of CIN3+ in hrHPV-positive women invited for routine screening.

Additional file 22 Supplementary Figure 6. LefSe analysis: relative abundances association with hrHPV status. Relative abundance counts of G. vaginalis (A), M. genomosp type 1 (B), S. amnii (C), S. sanguinegens (D), P. anaerobius (E), D. micraerophilus (F), A. vaginae (G), P. amnii (H), and P. buccalis (I) were found significantly over-represented in hrHPV positive women whereas Lactobacillus acidophilus (J) was enriched in hrHPV negative women.

Additional file 12 Supplementary Figure 3. CiRNAseq specificity towards Bifidobacterium and Gardnerella species. 16S rRNA-seq targets two variable regions (VRs) of the 16S rRNA gene using a forward and a reverse primer (e.g., V3 and V4). Alternatively, CiRNAseq targets five VRs of the 16S subunit using five singular smMIPs to differentiate species of B.longum and G. vaginalis. There is a high percentage of similarity (>90%) when comparing the V3-V4 regions of B. longum and G. vaginalis, which, if it is not appropriately amplified, could result in misidentification. In contrast, the CiRNAseq ROIs for both species have different levels of identity. Two out of five ROIs also possess a high percentage of similarity (>90%), with both amplifying the V7 and V8 VRs, and needed to identify these microbes at the family level. The rest remaining three out of five ROIs share less than 45% of similarity, which endorses the resolution and specificity of CiRNAseq in detecting both species. The color red represents similarity in sequences, while the color black represents no similarity.

Additional file 6 Supplementary Figure 1. In silico validation of CiRNAseq specificity towards cervicovaginal microbial species. CiRNAseq targets multiple 16S rRNA gene VRs e.g. V5 ��� V9 for microbiome profiling. For reads assigning and species annotation, the method has a threshold of 95% of similarity between sequences and reference ROIs (A). Phylogenetic analyses of the 16S RNA gene for the species Anaerococcus vaginalis, Anaerococcus tetradius, Peptostreptococcus anaerobius, Gardnerella vaginalis, and Prevotella buccalis shows the similarity between the 16S rRNA genomes (B). Alignment analyses of the ROIs from the closest related species to the least related species, according to (B), demonstrate the specificity of CiRNAseq. For sequencing A. vaginalis and A. tetradius the technique uses the same set of five smMIPs, but two out of five ROIs exhibit

Additional file 9. In silico alignment analyses (.pdf). Results obtained with Clustal Omega v1.2.4 for Supplementary Figures 1 and 3.

Additional file 21 Supplementary Figure 5. Average analysis of hrHPV cohort. Average analysis of hrHPV negative versus hrHPV positive with CIN2+ demonstrates a strong association of absence and presence of particular microbes. L. acidophilus, L. jensenii and L. crispatus were highly present in hrHPV negative women, but the microbiome seemingly changes to a diverse-anaerobic microbiota in hrHPV positive women with CIN2+.

Offering self‐sampling for HPV testing improves the effectiveness of current cervical screening programs by increasing population coverage. Molecular markers directly applicable on self‐samples are needed to stratify HPV‐positive women at risk of cervical cancer (so‐called triage) and to avoid over‐referral and overtreatment. Deregulated microRNAs (miRNAs) have been implicated in the development of cervical cancer, and represent potential triage markers. However, it is unknown whether deregulated miRNA expression is reflected in self‐samples. Our study is the first to establish genome‐wide miRNA profiles in HPV‐positive self‐samples to identify miRNAs that can predict the presence of CIN3 and cervical cancer in self‐samples. Small RNA sequencing (sRNA‐Seq) was conducted to determine genome‐wide miRNA expression profiles in 74 HPV‐positive self‐samples of women with and without cervical precancer (CIN3). The optimal miRNA marker panel for CIN3 detection was determined by GRridge, a penalized method on logistic regression. Six miRNAs were validated by qPCR in 191 independent HPV‐positive self‐samples. Classification of sRNA‐Seq data yielded a 9‐miRNA marker panel with a combined area under the curve (AUC) of 0.89 for CIN3 detection. Validation by qPCR resulted in a combined AUC of 0.78 for CIN3+ detection. Our study shows that deregulated miRNA expression associated with CIN3 and cervical cancer development can be detected by sRNA‐Seq in HPV‐positive self‐samples. Validation by qPCR indicates that miRNA expression analysis offers a promising novel molecular triage strategy for CIN3 and cervical cancer detection applicable to self‐samples., What's new? MicroRNAs (miRNAs) are suspected of playing a role in cervical cancer development. They are also potential markers for the identification of human papillomavirus (HPV)‐infected women who are at risk of cervical cancer. Here, using small RNA sequencing of HPV‐positive self‐samples from women with and without cervical precancer (CIN3), the authors identify a miRNA signature consisting of multiple miRNAs that is strongly predictive of CIN3. Validation of this signature by qPCR revealed a good clinical performance for CIN3+ detection. The findings suggest that miRNA analysis is an effective means of CIN3+ prediction in HPV‐positive self‐samples obtained for cervical cancer screening.

Contains fulltext : 171209.pdf (Publisher’s version ) (Closed access) Pneumocystis jirovecii can cause life-threatening pneumonia following treatment for haematological malignancies or after HSCT. The mortality rate of P. jirovecii pneumonia (PCP) in these patients is 30%-60%, especially after HSCT. The clinical presentation of PCP in haematology differs from that associated with HIV infection, with the disease being acute and more often severe, having a lower fungal burden and being more frequently linked to treatment with corticosteroids. Most cases occur in patients not receiving adequate prophylaxis. The development of new therapies, including targeted treatments and monoclonal antibodies in various haematological diseases, justifies constant vigilance in order to identify new at-risk populations and give prophylaxis accordingly. The fifth and sixth European Conferences on Infections in Leukaemia (ECIL-5 and ECIL-6) aimed to review risk factors for PCP in haematology patients and to establish evidence-based recommendations for PCP diagnosis, prophylaxis and treatment. This article focuses on the magnitude of the problem, the main differences in clinical presentation between haematology patients and other immunocompromised populations, especially HIV-infected patients, and the main risk factors.

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal qPCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicentre and monocentre evaluation of PCP qPCR assays was performed. For the multicentre study, 16 reference laboratories from eight different countries, performing 20 assays analysed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads respectively). The monocentre study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, Reverse Transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation

Offering self-sampling for HPV testing improves the effectiveness of current cervical screening programs by increasing population coverage. Molecular markers directly applicable on self-samples are needed to stratify HPV-positive women at risk of cervical cancer (so-called triage) and to avoid over-referral and overtreatment. Deregulated microRNAs (miRNAs) have been implicated in the development of cervical cancer, and represent potential triage markers. However, it is unknown whether deregulated miRNA expression is reflected in self-samples. This study is the first to establish genome-wide miRNA profiles in HPV-positive self-samples to identify miRNAs that can predict the presence of CIN3 and cervical cancer in self-samples. Small RNA sequencing (sRNA-Seq) was conducted to determine genome-wide miRNA expression profiles in 74 HPV-positive self-samples of women with and without cervical precancer (CIN3). The optimal miRNA marker panel for CIN3 detection was determined by GRidge, a penalized method on logistic regression. Six miRNAs were validated by qPCR in 191 independent HPV-positive self-samples. Classification of sRNA-Seq data yielded a 9-miRNA marker panel with a combined Area Under the Curve (AUC) of 0.89 for CIN3 detection. Validation by qPCR resulted in a combined AUC of 0.78 for CIN3+ detection. This study shows that deregulated miRNA expression associated with CIN3 and cervical cancer development can be detected by sRNA-Seq in HPV-positive self-samples. Validation by qPCR indicates that miRNA expression analysis offers a promising novel molecular triage strategy for CIN3 and cervical cancer detection applicable to self-samples. This article is protected by copyright. All rights reserved.

Figure S3. The expression levels of eight selected genes, verified by RT-PCR. The values were normalized to the actin expression, and error bars represent standard deviation of the mean. (PDF 297kb)

Figure S1. Dose-response curves of the three A. fumigatus strains upon exposure to increasing concentrations of itraconazole, ranging from 0.016â mg/l to 8â mg/l. (PDF 36 kb)

The mutant strains used for forming the heterokaryons and diploids in Figure 3 and 5.

Table S1. Mapping results and coverage calculations. (DOCX 79 kb)

The mutant strains used for forming the heterokaryons and diploids in Figure 3 and 5.

Five Patient characteristics including the underlying disease, the number of isolates collected and resistance switch during the treatment.

Figure S2. PCA plot of all samples, normalized by a regularized log transformation. (PDF 113 kb)

Additional file 1. Detailed description of methods for calculating results. Table A1. Age groupings used in analysis by programme type. Table A2. Calculation of the indicators shown in Figure 2 for participation, referral and detection within the new hrHPV-based screening programme, 2017 cohort. Table A3. Calculation of the indicators for participation, referral and detection in the old cytology-based screening programme, cohort 2015, and within the new hrHPV-based screening programme, 2017 cohort.

Contains fulltext : 172373.pdf (Publisher’s version ) (Closed access) The 5th European Conference on Infections in Leukaemia (ECIL-5) meeting aimed to establish evidence-based recommendations for the prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in non-HIV-infected patients with an underlying haematological condition, including allogeneic HSCT recipients. Recommendations were based on the grading system of the IDSA. Trimethoprim/sulfamethoxazole given 2-3 times weekly is the drug of choice for the primary prophylaxis of PCP in adults ( A-II: ) and children ( A-I: ) and should be given during the entire period at risk. Recent data indicate that children may benefit equally from a once-weekly regimen ( B-II: ). All other drugs, including pentamidine, atovaquone and dapsone, are considered second-line alternatives when trimethoprim/sulfamethoxazole is poorly tolerated or contraindicated. The main indications of PCP prophylaxis are ALL, allogeneic HSCT, treatment with alemtuzumab, fludarabine/cyclophosphamide/rituximab combinations, >4 weeks of treatment with corticosteroids and well-defined primary immune deficiencies in children. Additional indications are proposed depending on the treatment regimen.

Item does not contain fulltext The Fifth European Conference on Infections in Leukaemia (ECIL-5) convened a meeting to establish evidence-based recommendations for using tests to diagnose Pneumocystis jirovecii pneumonia (PCP) in adult patients with haematological malignancies. Immunofluorescence assays are recommended as the most sensitive microscopic method (recommendation A-II: ). Real-time PCR is recommended for the routine diagnosis of PCP ( A-II: ). Bronchoalveolar lavage (BAL) fluid is recommended as the best specimen as it yields good negative predictive value ( A-II: ). Non-invasive specimens can be suitable alternatives ( B-II: ), acknowledging that PCP cannot be ruled out in case of a negative PCR result ( A-II: ). Detecting beta-d-glucan in serum can contribute to the diagnosis but not the follow-up of PCP ( A-II: ). A negative serum beta-d-glucan result can exclude PCP in a patient at risk ( A-II: ), whereas a positive test result may indicate other fungal infections. Genotyping using multilocus sequence markers can be used to investigate suspected outbreaks ( A-II: ). The routine detection of dihydropteroate synthase mutations in cases of treatment failure is not recommended ( B-II: ) since these mutations do not affect response to high-dose co-trimoxazole. The clinical utility of these diagnostic tests for the early management of PCP should be further assessed in prospective, randomized interventional studies.

Pneumocystis jirovecii is an airborne human-specific ascomycetous fungus responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, affecting > 500,000 patients per year (www.gaffi.org). The understanding of its epidemiology is limited by the lack of standardised culture. Recent genotyping data suggests a limited genetic diversity of P. jirovecii. The objective of the study was to assess the diversity of P. jirovecii across European hospitals and analyse P. jirovecii diversity in respect to clinical data obtained from the patients. Genotyping was performed using six already validated short tandem repeat (STR) markers on 249 samples (median: 17 per centre interquartile range [11 − 20]) from PCP patients of 16 European centres. Mixtures of STR markers (i.e., ≥ 2 alleles for ≥ 1 locus) were detected in 67.6% (interquartile range [61.4; 76.5]) of the samples. Mixture was significantly associated with the underlying disease of the patient, with an increased proportion in HIV patients (78.3%) and a decreased proportion in renal transplant recipients (33.3%) (p

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Methods, supplementary figures, supplementary tables

Background The purpose of this retrospective study was to identify prognostic factors in a cohort of patients with oropharyngeal carcinoma treated with intensity-modulated radiotherapy (IMRT). Methods Medical records of 142 patients treated with (chemo)radiotherapy between September 2005 and September 2011 were reviewed and the human papillomavirus (HPV) status was determined by polymerase chain reaction (PCR) analysis. Potential prognostic factors for 3-year locoregional control and overall survival (OS) were evaluated. Results HPV-positive patients (n = 82) had locoregional control and OS of 78% and 79%, respectively. Significant prognostic factors on multivariate analysis were smoking (p = .03) for locoregional control and OS, and comorbidity (p = .04) for OS. Further stratification was done according to smoking behavior in HPV-positive patients. Locoregional control in current smokers was 67% compared to 86% in never smokers and former smokers, respectively (p = .02). Conclusion Smoking was the only modifiable prognostic factor in HPV-positive patients. Therefore, active stop-smoking programs must be integrated in the routine management of patients to maximize treatment results.

The European Aspergillus PCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated “ in vivo ” specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and β- d -glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and β- d -glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and β- d -glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and β- d -glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.

Contains fulltext : 95722.pdf (Publisher’s version ) (Open Access) The prevalence and spread of azole resistance in clinical Aspergillus fumigatus isolates in the Netherlands are currently unknown. Therefore, we performed a prospective nationwide multicenter surveillance study to determine the effects of resistance on patient management strategies and public health. From June 2007 through January 2009, all clinical Aspergillus spp. isolates were screened for itraconazole resistance. In total, 2,062 isolates from 1,385 patients were screened; the prevalence of itraconazole resistance in A. fumigatus in our patient cohort was 5.3% (range 0.8%-9.5%). Patients with a hematologic or oncologic disease were more likely to harbor an azole-resistant isolate than were other patient groups (p

Candida türleri, ökaryot fırsatçı patojenler olup hastane enfeksiyonu etkenleri arasında dünyada ön sıralarda yer almaktadır. Amerika'da hastanelerde gelişen nozokomiyal kan dolaşımı enfeksiyonlarının dördüncü sıklıktaki etkeni Candida türleridir. Avrupa'da kandidemi oranı ise her 100.000 kişide 1.2 ile 11 arasında değişmektedir. Candida'ların 200'den fazla türü bulunur, bunlar arasında dissemine kandidoz ile invazif olmayan deri ve mukoza kandidozlarının en sık nedeni Candida albicans'tır. Bununla birlikte, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae, C. dubliniensis ve C. guilliermondii gibi albicans-dışı Candida türlerine bağlı kandidoz insidansı artmaktadır. Candida enfeksiyonunun hangi klinik şekilde sonlanacağı primer olarak konak savunması tarafından belirlenir. Klinik tablolar, deri ve mukoza kandidozları ile derin yerleşimli enfeksiyonlar olmak üzere iki gruba ayrılır. Deri ve mukoza enfeksiyonları içerisinde pamukçuk, Candida özefajiti, özefagus-dışı gastrointestinal kandidoz, Candida vaginiti ve deri kandidozu yer alır. Derin yerleşimli enfeksiyonlar; kronik dissemine kandidoz (hepatosplenik kandidoz), kandidemi ve çeşitli organların kandidozunu içerir. Mantar, invazif enfeksiyon oluşturmasına yardımcı olabilecek olan, proteinaz ve lipaz gibi enzimleri salgılama yeteneğindedir. Ancak, bu enzimlerin klinik açıdan önemi henüz aydınlatılamamıştır. Amerikan Enfeksiyon Hastalıkları Derneğinin Mikoz Çalışma Grubu (IDSA) ve Avrupa Klinik Mikrobiyoloji ve Enfeksiyon Hastalıkları Derneğinin Mantar Çalışma Grubu (ESCMID), farklı hasta gruplarında invazif kandidozun tedavisi için pratik kılavuzlar yayınlamışlardır. Vurgulanması gerekli bir diğer hususda, tedavi için önemli ölçütlerden birisinin de antifungal ilaç direnci olduğudur. Candida türleri, ilaçların hücre içerisine birikimini azaltan dışa atım pompalarının salınımı ile, antifungal hedef proteinlerinin yoğunluğunu ve yapısını değiştirerek veya hücre zarındaki sterol bileşimini değiştirerek antifungal ilaçlara karşı direnç geliştirebilirler. Ayrıca, kandidozların gelişmesinde genel risk faktörlerinin (örnek: bağışıklık sisteminin baskılanması) rolü önemli olmasına rağmen, bu durum tüm Candida enfeksiyonlarının gelişimini açıklamak için yeterli değildir. Birçok araştırmada; genetik farklılıklar ile kandidoz riski artışı arasında bir ilişkinin olduğu, mukoza kandidozu ve sistemik kandidozu ayırt ettirici farklı genetik yapıların bulunduğu bildirilmiştir. Bu makalede, Candida cinsi maya mantarlarının neden olduğu enfeksiyonların epidemiyolojisi, tanısı, tedavisi, antifungal ilaç direnci ve konak genetik yatkınlıklarına ilişkin güncel bilgiler özetlenmektedir, Candida and Candidosis: Updates on Epidemiology, Diagnosis, Treatment, Antifungal Drug Resistance, and Host Genetic SusceptibilityCandida species are eukaryotic fungal pathogens known to be aetiological agents of opportunistic and nosocomial infections in humans worldwide. Candida spp. are the fourth most common cause of nosocomial bloodstream infections acquired in hospitals in the United States. In Europe, candidemia rates vary between 1.2 and 11 per 100.000 population. There are over 200 species of Candida yeasts; among them, Candida albicans is the most common cause of disseminated and non-invasive mucocutaneous diseases. However, the incidence of candidosis due to non-albicans Candida spp. is increasing, including: C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. lusitaniae, C. dubliniensis and C. guilliermondii. The clinical outcome of Candida infections is primarily determined by the host's defense status and can be divided into mucocutaneous infections and deep-seated infections. Mucocutaneous infections include thrush, Candida esophagitis, nonesophageal gastrointestinal candidosis, Candida vaginitis, and cutaneous candidosis syndromes. Deep-seated infections include chronic disseminated candidosis (hepatosplenic candidosis), candidemia, and candidosis of various organ systems. The fungus is capable of secreting proteinases and lipases that can assist invasion, although the clinical importance of these enzymes is not clear Both the Mycoses Study Group of the Infectious Diseases Society of America (IDSA) and the Infectious Diseases Group of the European Organisation for Research and Treatment of Cancer (EORTC) have published practice guidelines for the treatment of invasive candidosis in various patient populations. Of note, antifungal drug resistance was an important factor that needed to be considered for treatment. Candida spp. became resistant to antifungal agents due to the expression of efflux pumps that reduces drug accumulation, alteration of the structure or concentration of antifungal target proteins, and alteration of membrane sterol composition. Moreover, despite the important role played by general risk factors, i.e., an immunocompromised immune system, they did not explain all cases of infection. Several studies reported a link between genetic variation and an increased risk for Candida infections, with a different genetic pattern being discerned between mucosal and systemic candidosis. This article summarizes up-to-date information on the epidemiology, diagnosis, and treatment of infections caused by the genus Candida, as well as antifungal drug resistance and host genetic susceptibility in affected patients.

M. Lappalainen, A. Jääskeläinen ja T. Smura ovat työryhmän ESCV-ECDC EV-D68 Study Grp jäseniä. Background: In August and September 2014, unexpected clusters of enterovirus-D68 (EV-D68) infections associated with severe respiratory disease emerged from North-America. In September, the European Centre for Disease Prevention and Control (ECDC) asked European countries to strengthen respiratory sample screening for enterovirus detection and typing in cases with severe respiratory presentations. Objectives: To provide a detailed picture of EV-D68 epidemiology in Europe by conducting a retrospective and prospective laboratory analysis of clinical specimens. Study design: An initiative supported by the European Society for Clinical Virology (ESCV) and ECDC was launched to screen for EV-D68 in respiratory specimens between July 1st and December 1st 2014 in Europe and to sequence the VP1 region of detected viruses for phylogenetic analytic purposes. Results: Forty-two institutes, representing 51 laboratories from 17 European countries, analyzed 17,248 specimens yielding 389 EV-D68 positive samples (2.26%) in 14 countries. The proportion of positive samples ranged between 0 and 25% per country. These infections resulted primarily in mild respiratory disease, mainly detected in young children presenting with wheezing and in immuno-compromised adults. The viruses detected in Europe are genetically very similar to those of the North-American epidemic and the majority (83%) could be assigned to clade B. Except for 3 acute flaccid paralysis (AFP) cases, one death and limited ICU admissions, no severe cases were reported. Conclusions: The European study showed that EV-D68 circulated in Europe during summer and fall of 2014 with a moderate disease burden and different pathogenic profile compared to the North-American epidemic. (C) 2015 The Authors. Published by Elsevier B.V.

The clinically validated high-risk human papillomavirus (hrHPV) Hybrid Capture 2 (HC2) and GP5+/6+-PCR assays were analyzed on an Indicating FTA Elute cartridge (FTA cartridge). The FTA cartridge is a solid dry carrier that allows safe transport of cervical samples. FTA cartridge samples were compared with liquid-based samples for hrHPV and high-grade cervical intraepithelial neoplasia (CIN) detection. One cervical sample was collected in a liquid-based medium, and one was applied to the FTA cartridge. DNA was eluted directly from the FTA cartridge by a simple elution step. HC2 and GP5+/6+-PCR assays were performed on both the liquid-based and the FTA-eluted DNA of 88 women. Overall agreement between FTA and liquid-based samples for the presence of hrHPV was 90.9% with GP5+/6+-PCR and 77.3% with HC2. The sensitivity for high-grade CIN of hrHPV testing on the FTA cartridges was 84.6% with GP5+/6+-PCR and only 53.8% with HC2. By comparison, these sensitivities on liquid-based samples were 92.3% and 100% for GP5+/6+-PCR and HC2, respectively. Therefore, the FTA cartridge shows reasonably good overall agreement for hrHPV detection with liquid-based media when using GP5+/6+-PCR but not HC2 testing. Even with GP5+/6+-PCR, the FTA cartridge is not yet capable of detecting all high-grade CIN lesions.

Invasive aspergillosis remains an important complication of immunosuppressive treatment regimens in humans. Triazoles are increasingly used in the prevention and management of invasive aspergillosis. Voriconazole is first choice for the primary therapy of invasive aspergillosis, and posaconazole was shown to be highly effective in preventing invasive aspergillus disease when given to high-risk groups prophylactically. Resistance in moulds is uncommon in clinical medicine, but might occur in specific patient groups. It appears that azole resistance might evolve in patients that harbor a high number of reproducing Aspergillus and are treated with azoles for long period of time. These conditions are present in patients with aspergilloma or other cavitary lung diseases caused by Aspergillus species. Azole resistance has been described to emerge in this patient group. Recently azole resistance was also reported in patients with acute invasive aspergillosis, where the conditions for resistance development appear to be less that optimal: fungal proliferation through hyphal elongation and relative short treatment episodes. This might suggest that azole resistance is caused by exposure outside the patient, for instance in the environment. Molecular studies have shown that triazole resistance in A. fumigatus is associated with amino acid substitutions in the cyp51A protein. Alterations might result in different patterns of azole resistance, primarily of itraconazole, with varying reduced activity of other triazoles such as voriconazole and posaconazole. Azole resistance in A. fumigatus has been associated with treatment failure. At present clinical microbiology laboratories do not routinely perform in vitro susceptibility testing, but it appears to be appropriate to test A. fumigatus isolates at least in those isolates recovered from patients that fail to azole therapy.

The coxsackievirus B3 3A protein forms homodimers and plays important roles in both viral RNA (vRNA) replication and the viral inhibition of intracellular protein transport. The molecular determinants that are required for each of these functions are yet poorly understood. Based on the NMR structure of the closely related poliovirus 3A protein, a molecular model of the coxsackievirus B3 3A protein was constructed. Using this structural model, specific mutants were designed to study the structure-function relationship of 3A. The mutants were tested for their capacity to dimerize, support vRNA replication, and block protein transport. A hydrophobic interaction between the monomers and an intermolecular salt bridge were identified as major determinants required for dimerization. We show that dimerization is important for both efficient vRNA replication and inhibition of protein transport. In addition, determinants were identified that were not required for dimerization but that were essential for either one of the biological functions of 3A. The combination of the in silico and in vivo results obtained in this study provides important insights in both the structural and functional aspects of 3A.

Objective: Streptococcus pyogenes is the cause of the classical childbed fever and can occur in both sporadic and epidemic form. Once an outbreak is identified on a maternity ward it is not only necessary to place the patients in strict isolation but also identify to the source of the infection. Fast reliable typing methods can aid in infection control. Methods: An outbreak of puerperal fever due to S. pyogenes was analyzed by random amplified polymorphic DNA (RAPD) analysis. Results: Identical fingerprint patterns were found in isolates of 3 patients, the throat and infected finger of the delivering obstetrician, 2 of the physician's family members, and from the cervix of a woman who was examined by the physician 7 months after the outbreak. The outbreak was stopped after antimicrobial treatment of the physician and his family members. Conclusions: RAPD typing appeared to be a fast and reliable tool for epidemiological studies of S. pyogenes and is probably more efficient in strain differentiation than classical M and T serotyping.

Additional file 5 Supplementary Table 2. E. coli growth experiment. T: time; TP: time point of analyses; OD: optical density; RNAc: RNA concentration in ng/uL; URC: unique read counts; R1: replicate 1; R2: replicate 2; URCm: mean of URC from R1 and R2.

Additional file 4. Supplementary Table 1. Bacterial species used for in vitro experiments. MMB: Department of Medical Microbiology, Radboudumc.

Additional file 2. List of smMIPs and sequences used for CiRNAseq profiling of the cervicovaginal microbiome (.pdf). List of designed 30 smMIPs with ligation and extension arms that target 434 relevant microbes in the cervicovaginal microbiome at the DNA and RNA levels using CiRNAseq.

Additional file 5 Supplementary Table 2. E. coli growth experiment. T: time; TP: time point of analyses; OD: optical density; RNAc: RNA concentration in ng/uL; URC: unique read counts; R1: replicate 1; R2: replicate 2; URCm: mean of URC from R1 and R2.

Additional file 16 Supplementary Figure 4. Pearson���s correlation for Lactobacillus, Gardnerella, Atopobium and Megasphaera. Pearson���s positive correlation obtained from comparing the detection of Lactobacillus (A) (r = 0.9605, p = 0.0006), Gardnerella (B) (r = 0.9384, p = 0.0018), Atopobium (C) (r = 0.9255, p = 0.0028), and Megasphaera (r = 0.8344, p = 0.0196) using both CiRNAseq and 16S rRNA-seq corroborates the specificity and sensitivity of CiRNAseq.

Additional file 4. Supplementary Table 1. Bacterial species used for in vitro experiments. MMB: Department of Medical Microbiology, Radboudumc.

Additional file 11 Supplementary Figure 2. E. coli growth experiment. A Two replicates of the samples subjected to CiRNAseq shows the reproducibility of the technique by the number of unique read counts (URC) obtained in each replicate. B Mean of the replicates��� URC. C OD and RNA concentrations analyzed in time points five (growth), eight (autoclaved), and nine (antibiotic) were comparable with each other. However, as expected, the E. coli growth sample from time point eight had no URC after autoclavation, while the sample treated with cefoxitin had low URC, suggesting inhibition of bacterial metabolic activities. OD and RNA concentrations were measured before autoclavation and antibiotic treatment. T: time in hours; OD: optical density; URC: unique read counts; ng/��L: RNA concentration. *, p

Additional file 11 Supplementary Figure 2. E. coli growth experiment. A Two replicates of the samples subjected to CiRNAseq shows the reproducibility of the technique by the number of unique read counts (URC) obtained in each replicate. B Mean of the replicates��� URC. C OD and RNA concentrations analyzed in time points five (growth), eight (autoclaved), and nine (antibiotic) were comparable with each other. However, as expected, the E. coli growth sample from time point eight had no URC after autoclavation, while the sample treated with cefoxitin had low URC, suggesting inhibition of bacterial metabolic activities. OD and RNA concentrations were measured before autoclavation and antibiotic treatment. T: time in hours; OD: optical density; URC: unique read counts; ng/��L: RNA concentration. *, p

Additional file 2. List of smMIPs and sequences used for CiRNAseq profiling of the cervicovaginal microbiome (.pdf). List of designed 30 smMIPs with ligation and extension arms that target 434 relevant microbes in the cervicovaginal microbiome at the DNA and RNA levels using CiRNAseq.