QIAamp DNA Mini Kit Research Articles

Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples

Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×101CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×103CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.

2 Next Generation Sequencing genotyping project in Spanish cystic fibrosis patients with uncharacterized CFTR mutations

Objectives: The purpose of this work is to implement non-invasive prenatal diagnosis (NIPD) of cystic fibrosis, based on analysis of cellfree fetal DNA (cffDNA) present in maternal peripheral blood. NIPD of CF was performed by detection of the F508del mutation. Methods: cffDNA occurs in plasma only in form of short fragments. By targeting the shorter DNA molecules, the fractional concentration of cffDNA is enriched. cffDNA was isolated from maternal plasma by the QIAamp DNA Mini Kit and Clean Circulating DNA Kit. Quality of the cffDNA isolation was evaluated by automate capillary electrophoresis on Fragment AnalyzerTM. For confirmation of cffDNA in a sample, we evaluated three approaches: fetal gender determination, comparison of STRs in maternal and fetal genome, and methylation analysis of the maspin gene promoter. In cooperation with TIBMolBiol, we designed HRM-based LightSNiP assays to detect the F508del. PCR amplicons needed to be shorter than 200bp to be able to analyze short fragments of cffDNA. Real-Time PCR is performed by using the DyNamo ColorFlash Probe qPCR Kit, Xceed qPCR Probe 2x Mix No-ROX and SensiFASTTM Probe No-ROX Kit. Results: Based on our results, we concluded that the cffDNA isolation performed by the evaluated kits does not differ in cffDNA yield, but the Clean Circulating DNA Kit separates the larger fragments (predominantly maternal) more effectively. We concluded that there is no significant difference between performances of three Real-Time PCR mastermixes used. Conclusion: It is currently possible to detect the F508del mutation in cffDNA samples; and analysis of more CF mutations for NIPD of CF should be implemented in the future.

PWE-102 Elucidating The Role of Non JEJUNI/Coli-Campylobacter in The Development of Colorectal Cancer Utilising Comparative Genomics to Study Their Pathogenic Potential

Introduction Emerging evidence links the gut microbiota to colonic inflammation and colorectal cancer (CRC). Studies have shown over-representation of oral bacterial species including Campylobacter spp. in CRC tissue and also njc-Campylobacter are over-represented in IBD wherein normal homeostasis has been lost. Understanding the role of njc-Campylobacter as an aetiological agent in CRC and elucidating their pathogenicity characteristics is of immense clinical importance. Aim To perform whole genome sequencing (WGS) of clinically derived njc-Campylobacter to compare/identify virulence traits potentially linked to disease presentation. Methods WGS of 21 unique clinically derived Campylobacter isolates was performed (7 Campylobacter showae, 3 Campylobacter ureolyticus and 11 Campylobacter concisus) from biopsy/faecal samples of subjects with malignancy, acute enteritis and IBD. DNA was extracted using the QIAamp DNA Mini Kit. Libraries were prepared using Illumina Nextera XT DNA Library Prep Kit and Nextera XT Index Kit v2, multiplexed, and paired-end sequenced on the Illumina MiSeq. De novo read assembly used the A5-miseq pipeline with QUAST. Genome annotation was performed using the rapid prokaryotic genome annotation tool Prokka. Results Genome assembly of C. showae strains resulted in 8 to 59 contigs per strain (mean number = 21; average genome size 2,199,446 nucleotides); C. concisus isolates had an average contig number of 72 and average genome size of 1,982,586 nucleotides. C. ureolyticus strains average contig number was 134 and average genome size of 1,608,008 nucleotides. Through Prokka annotation, the average number of coding sequences was 2215 for C. showae isolates (range 2050–2368) with function predicted for ~71.3%. For C. concisus the average number of coding sequences was 1985 (range 1833–2087) with function predicted for ~ 71.9%. C. ureolyticus had a lower number of coding sequences (average 1613) with function predicted for ~72.6%. We focused on the VirB4/D10 operon homologs, which are components of the Type IV secretion system. Contrary to previously published sequence data from oral strains, the majority of colonic C. showae isolated and 2 C. concisus strains possessed almost complete T4SS systems and additional virulence traits, including the exotoxin 9 gene. Conclusion The findings clearly demonstrate genetic differences among colonic Campylobacter isolates as compared to oral strains and sheds light on the pathogenic potential of these emerging colonic pathogens. The findings also further highlight the need for further sequencing studies to be undertaken. Disclosure of Interest None Declared

Prevalence of Legionella among pneumonia patients and environmental water samples in an Egyptian University Hospital

Abstract Objectives: to diagnose Legionella infection in community- acquired pneumonia (CAP) cases admitted to chest department of Zagazig University Hospital and in hospital- acquired pneumonia (HAP) patients hospitalized in the emergency and surgery ICU, to determine incidence of these disease condition and to identify risk factors. Another objective was to determine the occurrence of Legionella genus in the water of these departments. Subjects and methods: one hundred pneumonic patients; 50 patients with CAP and 50 with HAP were the subject of this laboratory-based, comparative cross sectional study. Full clinical history and lower respiratory tract specimens were collected from each patient. Water samples were taken from 25 water outlets. DNA was extracted by QIAamp DNA Mini Kit, and real time PCR amplification of 16s r-RNA gene was used for diagnosis of Legionella genus. Risk factors were analyzed by logistic regression analysis. Results: Legionella genus was identified in eight out of 50 patients of CAP (16%), and ten out of 50 patients with HAP (20%). As regards CAP, Legionella was prevalent in old- age, smoker males, with diabetes mellitus (DM) and/or chronic obstructive pulmonary disease (COPD). Gastrointestinal tract (GIT) and neurological manifestations were the main presentations. Seventy-five percent needed ICU admission. Concerning HAP, hospitalization for more than ten days and having a stroke or head trauma were significant risk factors. Ten out of the 25 water samples tested were positive for Legionella genus; seven samples were from the chest department and three were from emergency ICU. No water-contamination with Legionella was found in the surgery ICU. Discussion: diagnosis of Legionella should be considered for both CAP and HAP in our locality. Periodic surveillance for detection of that genu s with subsequent disinfection of water sources when indicated should be carried out.

A Comparative Oncology Analysis of Canine T-Cell Lymphoma (TCL): Immunohistochemical Study and Next Generation Sequencing for Protein Analysis and Genomic Target Discovery

Background: TCLs are an uncommon, heterogeneous group of neoplasms with no consensus on optimal treatment and human 5-year survival rates <20-30%. The canine provides a potentially attractive model to study TCL in part given their spontaneously occurring cancers, intact immune system, and phylogenetic resemblance to humans. Furthermore, approximately 1/3 of all lymphomas in canine are TCL (cTCL). Previous research from our group (ASH 2014, #74755) identified 118 differentially expressed genes by RNA seq analyses comparing canine PTCL with normal canine lymph node, and PI3K, GATA3, GRB2 and PPARG as candidate biomarker genes by canonical pathway and network analyses. We aimed to further interrogate the canine as a model by histologic review and detailed genomic examination of cTCL.Methods: We evaluated de novo cTCL with immunohistochemistry (IHC) and next generation DNA sequencing for a priori genes. IHC on canine TCL was evaluated with human antibodies against CD5, CD79a, Ki67, CD3, CD4, CD8 and CD30. Canine DNA was extracted from 14 fresh frozen tissue samples and 2 paraffin embedded blocks using the QIAamp DNA Mini Kit. Utilizing the human Cancer Hotspot Panel v2 (hCHPv2), a custom expanded panel of 68 genes actively expressed in lymphoma tumor cells was created to screen cTCL for mutations. COSMIC database and PubMed was used to identify common variants expected to be present. Targets from the hCHPv2 were converted from the human genome (hg19) to positions in the canine genome (canFam3) using liftOver (UCSC Genome Browser). Following targeted amplification using the custom canine library, DNA sequencing was performed with the Ion Personal Genome Machine resulting in 4,527,638 total reads with an average length of 229 bases and 708x coverage per sample.Results: For IHC, we examined 10 primary cTCL cases utilizing human antibodies. The cTCL cases staining patterns included: 100% were CD79a negative; 80% were CD5+; Ki67 was variable; while the remaining multiple markers did not react to human antibodies. We subsequently evaluated 16 primary cTCL tumor tissue samples using DNA sequencing. There were 331 unique variants and 1474 total variants; each sample had an average of 92 variants. The most prevalent coding consequences mutations were intron variations (68%), followed by synonymous (14%) and missense variations (9%) (Fig. 1A). The most prevalent mutations were found in ATM, KIT, ERBB4, TNFAIP3, and TET2 (Fig. 1B). ATM has been implicated as a tumor suppressor and mutations have been described on a case basis in human thymic and mantle cell lymphomas, however, not in TCL. Furthermore, the majority of ATM variants identified in our analysis were non-coding or synonymous. In ATM, there were 22 mutations with 17 found as introns. The 5 coding mutations had only one of which was missense (M1758T), the others were silent (Fig. 1C). Conversely, the most prevalent variants found in the coding region were found in SMO, TP53, TNFAIP3, and TET2. Of all coding variants, 15 missense variants in TNFAIP3, JAK2, MYC, MET, SMO, DNMT3A, RB1, PIK3CA, TP53, and ERBB2 appeared to be deleterious through bioinformatics analysis. Additionally, a frameshift variant in CDH1 resulted in a 3 bp deletion that has not been described in dbSNP. Finally, mutations in KIT, TNFAIP3 and TET2 have been described in canine and human TCL with varying frequency.Conclusion: To the best of our knowledge, this represents one of the first genomic comparative oncology analyses conducted in TCL. Collectively, the DNA sequencing analysis in cTCL identified genomic similarities and novel mutations that may help unearth new oncogenic pathways in human TCL (e.g., ATM). Furthermore, the deleterious frameshift and missense mutations identified in this study as from TNFAIP3 and CDH1 are novel, of which has not been described in prior research. Continued investigation is needed towards the enhanced delineation of protein expression of cTCL and examination of the functional impact of genomic perturbations identified in cTCL in comparison to human TCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction.

BackgroundMitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substantial chloroplast DNA (cpDNA) and nuclear DNA (nDNA) and its end use efficiency can be reduced. Clearly, an effective mitochondria isolation method is warranted with wider applicability and with minimum contamination from cpDNA and nDNA.ResultsHere we reported an improved method for isolating mitochondria from dry wheat seeds and its extension to dead seeds, viable seeds, etiolated leaf tissue and several other plant species: oat, Arabidopsis, flax, and yellow mustard. The isolated mitochondria were successfully used to extract mtDNA with QIAamp DNA mini kit (Qiagen). The extracted mtDNA from the assayed samples of these species was intact in large quantity and showed little contamination from nDNA, cpDNA, RNA, and proteins. The mtDNA extracted from dead wheat seeds was also substantial, but more degraded and less intact when compared to those from viable seeds and other tissues.ConclusionThe improved method was successfully applied to isolate mitochondria and extract mtDNA from several different tissues and plant species. The major advance in the improvement lies in its wider application with the same mitochondria extraction medium to different tissues and species. The improvement is significant, as it helps to widen the scope of future plant mitochondria research.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0099-x) contains supplementary material, which is available to authorized users.

Comparison of Automated and Manual DNA Isolation Methods for DNA Methylation Analysis of Biopsy, Fresh Frozen, and Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples.

Automated DNA isolation can decrease hands-on time in routine pathology. Our aim was to apply automated DNA isolation and perform DNA methylation analyses. DNA isolation was performed manually from fresh frozen (CRC = 10, normal = 10) specimens and colonic biopsies (CRC = 10, healthy = 10) with QIAamp DNA Mini Kit and from FFPE blocks (CRC = 10, normal = 10) with QIAamp DNA FFPET Kit. Automated DNA isolation was performed with MagNA Pure DNA and Viral NA SV kit on MagNA Pure 96 system. DNA methylation of MAL, SFRP1, and SFRP2 were analyzed with methylation-specific high-resolution melting analysis. Yield of automatically isolated samples was equal in fresh frozens and significantly lower compared to manually isolated biopsy and FFPE samples. OD260/280 of fresh frozen and biopsy samples were similar after both isolations, automated isolation resulted in lower purity in FFPE samples. Both protocols resulted in similar OD260/230 from fresh frozens, automated isolation method was superior in biopsies and manual protocol in FFPE samples. DNA methylation of biopsies, fresh frozen samples were highly similar after both methods, results of automatically and manually isolated FFPE samples were different. Automated DNA isolation from fresh frozen samples can be suitable for high-throughput laboratories.

Case-control based study between polymorphisms in the adiponectin gene and polycystic ovary syndrome

To investigate the frequeney of four single nucleotide polymorphism (SNP) sites (rs17300539, rs12495941, rs2241766 and rs1501299) of adiponectin gene (ADIPOQ) and to elucidate its role in the pathogenesis of polycystic ovary syndrome (PCOS). A total of 207 women with PCOS and 192 controls were recruited. Four ml whole-blood samples were collected in tubes containing ethylene diamine tetraacetic acid (EDTA) by peripheral venous puncture. Genomic DNA was extracted using a QIAamp DNA mini kit. Four SNP sites (rs17300539, rs12495941, rs2241766 and rs1501299) of ADIPOQ were amplified by PCR and then directly sequenced to screen variants. (1) The genotype frequencies of AA of rs17300539 in PCOS was significantly higher than controls [57.5% (119/207) versus 48.4% (93/192), P<0.05]. The genotype frequencies of AA of rs1501299 in PCOS was significantly lower than controls [4.8% (10/207) versus 11.5% (22/192), P<0.05]. While no significant differences were found in rs2241766 and rs12495941 (P>0.05). (2) The allele A of rs17300539 [75.8% (314/414)] and allele C frequeneies of rs1501299 [76.3% (316/414)] in PCOS were significantly higher than controls [67.7% (260/384), 69.0% (265/384), respectively; all P<0.05]. While no significant differences were found in rs2241766 and rs12495941 (P>0.05). (3) Further analysis we found rs17300539 AA genotypes had an increased risk for PCOS compared with GG genotype (OR=2.670, P=0.009), rs1501299 CC genotype had an increased risk for PCOS compared with AA genotypes (OR=2.756, P=0.012); and the difference remained significantly after adjustment for age, testosterone and body mass index (P<0.05). No significant differences were observed in genotype and allele frequencies between PCOS and controls for rs2241766 and rs12495941. However, we observed an association between rs17300539, rs1501299 and PCOS. rs17300539 and rs1501299 of ADIPOQ perhaps are the susceptibility gene locus of PCOS.

P77: Clonal evolution of breast tumor during neoadjuvant chemotherapy and metastasis

Background There are numerous evidences suggesting that tumor evolution follows the laws of Darwinian evolution, whereby individual tumor cell clones have private genetic aberrations, including chromosomal abnormalities. The combined effect of genetic instability and differential selective pressures of the microenvironment and chemotherapy can result in the creation of new tumor clones (Navin et al., 2011; Ng et al., 2012). The aim of this study is to show breast tumor clonal evolution during neoadjuvant chemotherapy (NAC) using microarray analysis. Material and methods Breast cancer patients ( n = 26) with stage IIA to IIIC (T1-4N0-3M0), were treated with NAC (FAC or CAX regimens). DNA was extracted from 26 samples of tumor tissue derived before or after NAC using QIAamp DNA mini Kit (Qiagen, Germany). Copy Number Aberrations (CNA, deletions and amplifications, or Loss and Gain, respectively) and number of mutant clones were detected in pre- and post-NAC tumor samples using the high density microarray platform Affymetrix (USA) CytoScanTM HD Array. This study was approved by Tomsk Cancer Research Institute review board. Results We have revealed that 19% (5/26) of patients during the NAC showed the decrease in the number of mutant clones and CNA frequency right up to their complete elimination (genetic regression) at one case. In 7 (27%) cases chemotherapy had no any effect on number of mutant clones and the frequency of CNA in tumor. In the tumors of 10 patients the elimination of some mutant clones as well as the formation of new clones with deleted genetic material occurred under the influence of chemotherapy. 6 patients have demonstrated the appearance of new tumor clones with gene amplifications which were associated with the development of metastases in 83% of cases (5/6). All other patients ( n = 21) who has not acquired the new tumor clones with Gain function mutation after NAC did not manifest distant metastasis in 5-year follow-up (Kaplan–Meier, p = 0.00001 Log-rank test). Conclusion The first time evidence is presented that the formation of new tumor clones may occur during the NAC. Metastasis of breast cancer is associated with the appearance of new clones with DNA amplifications. Detection of these clones allow getting new prognostic factor in breast cancer. The study was supported by the Russian Foundation for Basic Research – Russia (Project 15-04-03091).

P120: Microarray research of allelic imbalance in breast cancers during neoadjuvant chemotherapy

Background For single nucleotide polymorphism – SNP (SNP – Single Nucleotide Polymorphism) is characterized by phenomenon of allelic imbalance (AI). The phenomenon of allelic imbalanced (AI) is typical of many genes in different malignancies. Allelic imbalanced may result allelic deletions (loss of one copy of the locus) or amplification of one allele, resulting in only a single allelic variant of SNP is determined in the tumor in the PCR. Phenomenon of AI in tumors of breast cancer (BC) is considered in one or more genes. The phenomenon of AI during chemotherapy (CT) and especially in the longer format of the gene had not previously been studied. Thus, the aim of this work was to study microarray imbalanced allele in mammary tumors during neoadjuvant chemotherapy (NCT). Materials and methods The study included 26 breast cancer patients with stage IIA – IIIC. The patients in the neoadjuvant mode received 2–4 courses of chemotherapy regimens FAC or CAX. DNA from 26 paired samples before treatment and operational samples were isolated by dialing QIAampDNA miniKit (Qiagen, Germany). Microarray analysis was performed on DNA chips of high density company Affymetrix (USA) CytoScanTM HD Array, which contains more than 750 thousand SNP. Microarray analysis was performed on SNP genotypes DNA tumor tissue before and after treatment for each patient and recorded as change occurring allelic imbalanced tumor tissue genotype (AA > AB > AB AA BB > AB > AB BB) during therapy. Results The frequency of the AI in breast tumor during NCT was highly variable (within 0.9–66.5%) of the studied SNP (6850 – 497,979 SNP). For each patient, frequency shift genotype (homozygous in heterozygous genotype, and vice versa) was calculated as a percentage of all the shifts. Changes in the wild or mutant heterozygous genotypes (AA or BB > AB) were combined into one group; the second group was the sum of the change in the heterozygous genotype homozygous wild genotype or the mutant (AB > AA or BB). We have found that the direction of the AI was significantly associated effect of NCT. In the group of patients with partial regression, the direction of AI change from homozygous to heterozygous genotype often occurs (AA or BB > AB) (9/14, 64%), whereas patients with no response to the NCT (with stabilization or progression) have the opposite effect. All these patients (12/12 cases) have the direction of the change of AI from heterozygous genotype to homozygous (AA or AB > BB) ( p = 0.00071). AI during chemotherapy at the level of the marked tendency (Log-rank test, p = 0.062) is associated with 5-year metastasis-free survival. Low metastasis-free survival rate is observed in patients with AI in the direction of the change from the heterozygous to homozygous genotype, while 100% survival is noted in patients with change from homozygous to heterozygous genotypes, and this imbalanced t allele is a favorable prognostic factor. Conclusion Allelic imbalanced in breast tumor during NCT phenomenon is massive and may affect up to 67% of SNP. AI may occur in the direction of change from homozygous to heterozygous genotype, and it is associated with a good response to treatment and 100% metastasis-free survival. Apparently, this can be explained by the fact that the change from homozygous genotypes to heterozygous occur due to partial destruction of tumor cells by chemotherapy, resulting in the increase of stromal elements. In contrast, AI in the direction change from heterozygous to homozygous genotype during NCT is associated with no response to chemotherapy due to metastasis and occurring of new mutant clones in the tumor.

Lack of Chlamydia-related bacteria among patients with community-acquired pneumonia.

The aetiology of community-acquired pneumonia is only determined in approximately 50% of cases [1]. Chlamydia and Chlamydia-related bacteria may be the agents of some pneumonia of unknown etiology. Indeed, C. psittaci and C. pneumoniae are well-established agents of pneumonia [2], whereas Parachlamydia acanthamoebae and Simkania negevensis are considered to be emerging agents of lung infections [3], [4]. However, routine cultures fail to detect these strict intracellular Chlamydiales, and broad-range eubacterial 16S rRNA PCRs (applied to lung biopsy or pleural fluid samples) also fail to detect Chlamydiales [5]. Moreover, several additional bacteria belonging to the Chlamydiales order might possibly be involved in lung infections, including Rhabdochlamydia spp. and Waddlia spp., as well as new lineages. To fully investigate the role of all members of the Chlamydiales in lung infections, we thus investigated by a pan-Chlamydiales PCR oropharyngeal samples taken from a total of 564 patients with moderately severe community-acquired pneumonia included in a prospective study [6]. Briefly, DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Then we performed in duplicate a homemade pan-Chlamydiales real-time PCR, as described elsewhere [7]. The results of all tests but one were negative. The positive sample, taken from a 79-year-old Swiss man, showed the presence of C. psittaci DNA. The patient was hospitalized for fever and cough. He complained of acute-onset cough, dyspnea on minimal exertion and fever. His medical history was notable for cerebrovascular disease, coronary artery disease and diabetes mellitus. There was no recent travel or documented exposure to animals. At admission the patient was disoriented, hypoxemic and febrile (38.3°C), with mild tachypnea and tachycardia. Chest examination revealed dullness on percussion and crackles at the left pulmonary base. He exhibited mild leukocytosis and elevated urea. Consolidation of the left lower lobe and a small pleural effusion were documented by chest X-ray. Intravenous cefuroxime and oral clarithromycin were administered. His condition rapidly improved, and the patient was transferred on the tenth day after admission to a rehabilitation facility. Two pairs of blood cultures, obtained before the administration of any antibiotics, remained sterile. Urine was negative for the presence of Legionella pneumophila and Streptococcus pneumoniae antigens. Normal oropharyngeal flora was recovered by culture from a sputum sample. Testing for the presence of C. pneumoniae and Mycoplasma pneumoniae by PCR on the oropharyngeal swab was also negative. Contrarily to what we expected, no Chlamydia-related bacteria have been detected by the pan-Chlamydiales PCR in the largest cohort published to date. This contrasts with previous work by Marrie et al. [8], who identified two seroconversions among 255 patients with community-acquired pneumonia requiring hospitalization (0.8%), and with the study by Casson et al. [9] documenting DNA of Parachlamydia acanthamoebae in 15% of nasopharyngeal swabs taken from children with a bronchiolitis. The difference may be due to the different study populations. Here, only adults with mild and moderate community-acquired pneumonia were enrolled and immunocompromised patients were excluded, whereas in previous studies P. acanthamoebae was mainly documented among immunosuppressed patients [8], [10] and children [7], [9]. The absence of S. negevensis DNA argues against a pathogenic role of Simkania, a still-controversial question [2], [3]. On the other hand, the documented C. psittaci infection underlines the importance of a complete diagnostic assessment, including C. psittaci, even in absence of history of exposure to birds. These data, taken together with the findings of our study, demonstrate that outside specific local conditions that may lead to cases clusters or outbreaks, different members of the Chlamydiales order, including C. pneumoniae, are not common agents of community-acquired pneumonia [11].

Stains of ejaculated pre and post-vasectomy: Purity and sufficient quantity of recovered DNA after 10 years of storage

Vasectomy is a widely used method of contraception. The absence of spermatozoa in the ejaculate of azoospermic individuals increases the difficulty of extracting DNA for analysis, especially if the biological samples are in the form of spots and stored for years. The aim of this study is to evaluate the possibility of obtaining autosomal and Y-STR profiles from stains containing the ejaculate of individuals pre and post-vasectomy, stored in cotton fabric for a period of 10 years. Samples from 28 (twenty eight) individuals: semen pre-vasectomy and seminal fluid (post-vasectomy) stains, stored from 2004 to 2014 at room temperature were used. Three cutouts of 1.5cm in diameter (punch) were taken from central region of each spot. It was used the QIAamp DNA Mini kit (Qiagen) to extract DNA with an adapted protocol followed by quantification by Nanodrop equipment. Genetic profiles were obtained using ESI17PowerPlexR (Promega). Capillary electrophoresis was performed on the ABI PRISM 3500XL DNA Analyzer. The DNA extraction was successful in all punch analyzed, and its concentration ranged from 1.1ng/uL to 23.3ng/uL in the pre- vasectomy and from 0.8ng/uL to 7ng/uL in postvasectomy punch. The samples had suitable purity, reflecting the presence of few contaminants. Currently, the techniques of DNA amplification are extremely sensitive and capable of assessing human DNA in small quantities generating partial or full profiles, trespassing situations such as long storage periods in cotton fabric. These data can be used as a benchmark for forensic analysis in cases of semen analysis.

Evaluation of suitable control genes for quantitative polymerase chain reaction analysis of maternal plasma cell-free DNA.

The content stability of commonly used control genes is considered to vary significantly in different independent experimental systems, either in the expression of RNA expression or in the level of DNA content. The present study aimed to examine a panel of six common control genes, including β‑globin (HBB), telomerase (TERT), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), albumin (ALB), β‑actin (ACTB) and T cell receptor γ (TRG), in order to evaluate and validate the most reliable control genes for quantitative polymerase chain reaction (qPCR) in investigations for the analysis of fetal‑derived DNA and maternal‑derived DNA in maternal plasma to enable non‑invasive prenatal assessment. Plasma DNA was extracted from the peripheral blood of 20 pregnant femals (gestational age, 18.67 ± 0.58 weeks) using a QIAamp DNA mini kit. Electrophoresis was performed to separate the fetal‑derived DNA and the maternal‑derived DNA at the 300bp position. qPCR was then performed, followed by geNorm‑, NormFinder‑ and BestKeeper‑based analyses to evaluated the content stabilities of the six candidate control genes in the fetal‑derived DNA and maternal‑derived DNA. The subsequent analysis of the experimental data revealed that HBB was expressed in the maternal‑ and fetal‑derived DNA together and in the maternal‑derived DNA alone. In addition, GAPDH in the fetal‑derived DNA enabled efficient normalization for qPCR investigations in the maternal plasma DNA.

Primary resistance to Hepatitis B Virus in the treatment-naive Patients

No: 1714 Presentation at ESCV 2015: Poster 2 Primary resistance to Hepatitis B Virus in the treatment-naive Patients M. Altindis1,∗, L. Demir1, I. Uslan2, Ozdemir Mehmet3, M. Baykan3, M. Kosecik4 1 Sakarya University School of Medicine, Dept of Clinical Microbiology, Sakarya, Turkey 2 Sakarya University School of Medicine, Dept of Gastroenterology, Sakarya, Turkey 3 Necmettin Erbakan Uni School of Medicine, Dept of clinical Microbiology, Konya, Turkey 4 Sakarya University School of Medicine, Dept of Pediatry, Sakarya, Turkey Background:Resistance to nucleos(t)ide analogues (NUC) is the main cause of NUC treatment failures leading to disease aggravation and complications. Our aim was to assess the prevalence of primary resistance to NUCs in a large cohort of treatment-naive patients infected with HBV by means of a highly sensitive method for the detection of minority viral populations. Methods: In this study, presence of resistance mutations in treatmentnaive individuals with chronic hepatitis B (CHB) was investigated in Sakarya University Hospital. A total of 59 patients (24 female, 35 male; age range: 19–66 years) diagnosed as CHB were enrolled in the study with informed consent from Gastroenterology, Sakarya anD Konya. All of the patients were negative for hepatitis C andD viruses and human immunodeficiency virus coinfections, and none had a history of interferon or NA treatment. HBV viral load(Montania® 4896, Bosphore® HBV Quantification Kit, Anatolia), HBV markers and hepatic enzymes in patients were determined via standardized commercial assays. For the detection of NA resistance mutations, a partial sequence of approximately 250 nucleotides, harboring the frequently-observed sites for NA resistance was amplified via nested PCR and characterized by direct sequencing of the amplicons(Bosphore® HBV Drug ResistanceMutation Sequencing Kit, Anatolia and Beckman Coulter CEQ 8000 Genetic Analysis System). The sequences were handled and interpreted for the presence ofmutations via various softwares and a web-based virtual phenotyping tool(CEQ 8000 Genetic Analysis System Version 9). Results: All circulating HBV strains were observed as genotype D(D1, D2, D3). At baseline, 59 patients harbored HBV resistanceassociated variants (RAV), generally in low proportions (0.3–20%). 26.9%, 9.7%, 1.6%, 1.6% of the patients displayed RAVs representing less than 1%, 1–5%, 5–10%, 10–20% of the viral quasispecies, respectively. 3 patients harbored RAVs that represented more than 20% of the quasispecies, including rtq215s, rtl91 and rtQ149K variant at 25%. None of the patients were found to harbor variants with both resistance and fitness mutations, which are generally selected on treatment at the sensitivity level of the technique. Conclusions: Primary HBV resistance to NUCs is frequent in treatment-naivepatients. These results further emphasize theneed for using first-line therapies including NUCs with a high barrier to resistance. http://dx.doi.org/10.1016/j.jcv.2015.07.273 Abstract No: 1725 Presentation at ESCV 2015: Poster 2 Development of an automated extraction system for Formalin-Fixed Paraffin Embedded (FFPE) tissue for the downstream detection of Human Papillomavirus (HPV) D. Guerendiain ∗, C. Moore, K. Cuschieri SHPVRL, NHS Lothian, Edinburgh, UK Background: Characterisation of FFPE biopsy material for HPV status is important for both epidemiological purposes and increasingly, for annotation of oropharyngeal squamous cell cancer (OPSCC). While manual extraction procedures for this application have been published–there is a lack of data on the development and performance of automated systems. The objective of this study was to develop and evaluate an automated extraction system for the detection of HPV nucleic acid using the NucliSENS® easyMAG®. Methods: A total of 60 samples were assessed: 30 FFPE sections related to high grade cervical intraepithelial neoplasia (CIN) lesions and 30 sections were derived from OPSCC. All samples had been extracted previously with manual silica columns (QIAamp DNA Mini Kit) and tested with the Optiplex HPV DNA assay (Diamex). Curled sections were obtained from each block with10 m thickness. Sampleswere extracted using the easyMAG® using a protocol adapted for FFPE material. DNA was tested using the Nanodrop® to evaluate the quantity and purity of DNA extracted. Agreement between the original HPV result associated with the manual extract and the results associatedwith the automated extract were assessed. Results: Nucleic acid yields ranged from 4.8 to 88ng/ l on extractsobtained fromtheeasyMAG® platform.At leastpartial concordance was observed in 93% in both cervical and OPSCC samples with an overall Kappa for HPV detection in the total sample (n=60) of 0.8049 (95% CI: 0.62–0.98). Specific kappas on HPV type-specific agreement will be presented. Conclusion:Our results indicate a high agreement between the easyMAG® and theQIAampDNAMiniKit® for the extractionofHPV DNA in FFPE samples derived from the oropharynx and the cervix This extraction method will reduce hands-on time – particularly for batch extraction. http://dx.doi.org/10.1016/j.jcv.2015.07.274

Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl–Neelsen-stained microscopic slides

Objective/backgroundThe diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl–Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit). MethodsDNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]⩾4), a low-codification-slide group (BI=1), a negative-slide group (BI=0), and a negative-control-slide group (BI=0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction. ResultsThis is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol–chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities. ConclusionThe ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques.

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

Efficiency of EGFR mutation analysis for small microdissected cytological specimens using multitech DNA extraction solution.

The microdissection method has greatly facilitated the isolation of pure cell populations for accurate analysis of mutations. However, the absence of coverslips in these preparations leads to poor resolution of cellular morphological features. In the current study, the authors developed the MultiTech DNA extraction solution to improve the visualization of cell morphology for microdissection and tested it for the preservation of morphological properties of cells, quality of DNA, and ability to detect mutations. A total of 121 cytological samples, including fine-needle aspirates, sputum, pleural fluid, and bronchial washings, were selected from hospital archives. DNA extracted from microdissected cells was evaluated by epidermal growth factor receptor (EGFR) mutation analysis using pyrosequencing, Sanger sequencing, and peptide nucleic acid (PNA)-mediated real-time polymerase chain reaction clamping. Morphological features of cells as well as DNA quality and quantity were analyzed in several cytological samples to assess the performance of the MultiTech DNA extraction solution. The results were compared with previous EGFR mutation tests. The MultiTech DNA extraction solution improved the morphology of archived stained cells before microdissection and provided a higher DNA yield than the commercial QIAamp DNA Mini Kit in samples containing a minimal number of cells (25-50 cells). The authors were able to detect identical EGFR mutations by using different analysis platforms and consistently identified these mutations in samples comprising as few as 25 microdissected cells. The MultiTech DNA extraction solution is a reliable medium that improves the resolution of cell morphology during microdissection. It was particularly useful in EGFR mutations of samples containing a small number of cells.

Evaluation of Ganciclovir Resistance in Cytomegalovirus Infection of Renal Transplant Recipients in Tehran

BackgroundHuman cytomegalovirus (CMV) infection is a major issue in solid organ transplant recipients. Although development of prophylaxis and preemptive procedures have presented significantly improved consequences in CMV infection, increasing incidence of antiviral resistance has raised virologists' concern. MethodsThe present study focused on kidney transplant recipients with high quantities of CMV load after antiviral therapy. We collected 5 mL blood from each of 58 patients. DNA extraction was performed with the use of the QIAamp DNA Mini kit (Qiagen), in accordance with the manufacturer's instructions. ResultsOur population study was 38% female and 62% male. CMV DNA was observed in 50 specimens (86%) with the range of 1.9 × 103 to 11 × 107 copies/mL serum. All of these patients had received ganciclovir for >3 months. Sequencing showed 18 mutations in 10 patients. Among these, 16 mutations were associated with Ul97 and the rest with Ul54 gene. Forty CMV-positive patients did not show any mutations. ConclusionsThe consequences of long-term ganciclovir resistance could not be determined.

Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing

Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform.

656 Elevated Serum Alkaline Phosphatase At Follow-Up Is Associated With Worse Outcomes in Primary Sclerosing Cholangitis

Background: Primary sclerosing cholangitis (PSC) is a progressive, chronic cholestatic liver disease characterized by inflammatory and fibrotic destruction of the intrahepatic and/or extrahepatic bile ducts. The pathogenesis of lesions in PSC are unknown. Bacterial components have been postulated to stimulate an aberrant immune response resulting in the perpetuation of the biliary inflammation seen in PSC. Alpha-hemolytic streptococci was associated with this disease in a previous study of bile samples from PSC patients, though more likely in those previously instrumented. Aims: To assess the microbial composition of bile collected via diagnostic endoscopic retrograde cholangiopancreatography (ERCP) in patients with PSC compared to controls using culture and qPCR.Methods: Bile was collected prospectively during routine ERCPs. A catheter was passed through the scope and bile was carefully aspirated upon entry in order to minimize contamination. Bacterial culture was performed using serial 10 fold dilutions of the sample in phosphate buffered saline (PBS) to achieve dilutions ranging from 10-1 to 10-3. A 0.1mL aliquot of the original sample and each dilution was plated onto enrichment media. The tubes were examined daily for growth for up to 5 days. We also extracted DNA from the bile using the tissue protocol in QIAamp DNA Mini Kit. Participants: 18 patients with primary sclerosis cholangitis (7 were ERCP naive) and 7 non-PSC patients Results: Of the 18 patients with PSC, alpha hemolytic streptococci was identified in 10 of the samples alone or in combination with staphylococcus. This was not related to having had a prior ERCP (p=.36). Eight of the PSC samples had no growth. Of the 7 non-PSC patients, alpha-hemolytic strep was found in three patients but only in combination with multiple other organisms. Among the control samples there was no one dominating species.11/18 PSC patients had concurrent inflammatory bowel disease. All 7 non-IBD patients had growth while only 3 of the IBD patients had growth. Samples from those without IBD were more likely to have growth (p=0.0025). 7/18 PSC patients had received antibiotics prior to the ERCP. Bacteria growth was not related to antibiotic use prior to ERCP (p=0.27). qPCR was done to confirm these finding which correlated when compared to a standard E.Coli curve. Conclusion: Alpha-hemolytic Streptococcus may play a role in the pathogenesis of primary sclerosing cholangitis as this was the dominant species cultured from bile samples of PSC patients. . While validating previous work, in this study alpha hemolytic strep was found in both ERCP naive patients and not. Alpha-hemolytic strep was not regularly seen in the non-PSC bile samples, and if present was in combination with multiple other species. This finding suggests antibiotics targeting alpha hemolytic streptococcus may be a useful therapy for PSC.