QIAamp DNA Mini Kit Research Articles

Biosynthetic gene cluster signature profiles of pathogenic Gram-negative bacteria isolated from Egyptian clinical settings.

Biosynthetic gene clusters (BGCs) are a subset of consecutive genes present within a variety of organisms to produce specialized metabolites (SMs). These SMs are becoming a cornerstone to produce multiple medications including antibacterial and anticancer agents. Natural products (NPs) also play a pivotal role in enhancing the virulence of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which represent a global health threat. We aimed to sequence and computationally analyze the BGCs present in 66 strains pertaining to three different ESKAPE pathogenic species: 21 A. baumannii, 28 K. pneumoniae, and 17 P. aeruginosa strains recovered from clinical settings in Egypt. DNA was extracted using QIAamp DNA Mini kit and Illumina NextSeq 550 was used for whole-genome sequencing. The sequences were quality-filtered by fastp and assembled by Unicycler. BGCs were detected by antiSMASH, BAGEL, GECCO, and PRISM, and aligned using Clinker. The highest abundance of BGCs was detected in P. aeruginosa (590), then K. pneumoniae (146) and the least in A. baumannii strains (133). P. aeruginosa isolates shared mostly the non-ribosomal peptide synthase (NRPS) type, K. pneumoniae isolates shared the ribosomally synthesized and post-translationally modified peptide-like (RiPP-like) type, while A. baumannii isolates shared the siderophore type. Most of the isolates harbored non-ribosomal peptide (NRP) BGCs with few K. pneumoniae isolates encoding polyketide BGCs. Sactipeptides and bottromycin BGCs were the most frequently detected RiPP clusters. We hypothesize that each species' BGC signature confers its virulence. Future experiments will link the detected clusters with their species and determine whether the encoded SMs are produced and cause their virulence. IMPORTANCE Our study analyzes the biosynthetic gene clusters (BGCs) present in 66 assemblies from clinical ESKAPE pathogen isolates pertaining to Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains. We report their sequencing and assembly followed by the analysis of their BGCs using several bioinformatics tools. We then focused on the most abundant BGC type in each species and we discussed their potential roles in the virulence of each species. This study is pivotal to further build on its experimental work that deciphers the role in virulence, possible antibacterial effects, and characterization of the encoded specialized metabolites (SMs). The study highlights the importance of studying the "harmful" BGCs and understanding the pathogenicity and virulence of those species, as well as possible benefits if the SMs were used as antibacterial agents. This could be the first study of its kind from Egypt and would shed light on BGCs from ESKAPE pathogens from Egypt.

351. INVESTIGATING THE IMPACT OF INDUCTION CHEMOTHERAPY FOR ESOPHAGEAL ADENOCARCINOMA ON THE GUT MICROBIOME

Abstract Background Esophageal adenocarcinoma (EAC) is a highly heterogeneous cancer with a current five-year survival rate of ~18%. To improve outcomes, understanding factors impacting treatment response is imperative. Gut microbiome signatures are one such factor and have been associated with response to immunotherapy and survival in several solid cancers. Thus, we aim to identify variations in gut microbiome signatures at different stages of treatment and elucidate the link of these signatures to treatment responses and patient outcomes. Methods Fecal samples were collected from 50 patients prior to induction and 18 during or post induction and stored at −20 ̊C before extracting DNA using the QIAamp DNA Mini Kit. Extracted DNA was quantified and 16S gene expression was confirmed by qPCR using primers targeting the V3-V4 region of the 16S gene, following which 16S rRNA sequencing was performed. FastQC, MultiQC and Cut Adapt were used for quality control of sequence data before assembly with vsearch. The resulting data was processed following the deblur pipeline. Taxonomy was assigned using Qiime2 classift-hybrid-vsearch-sklean function. Statistical comparison of diversity was conducted in R. Results Species diversity within individual samples, the alpha diversity, showed no significant difference between baseline and post-induction samples. Likewise, the comparison of diversity between samples, the beta diversity, showed no significant clustering on baseline compared to post-induction. On the genus level, there was no significant statistical difference between the normalized counts of individual genera shared between baseline and post-induction samples. Conclusion These preliminary results suggest that induction therapy has minimal impact upon species richness of the gut microbiome when compared to baseline as measured by fecal microbiome. Future directions of this project include examining the gut microbiome at further stages of treatment and investigating the microbiome of the tumour itself.

Human Brucellosis: An Observational Study From a Tertiary Care Centre in North India.

The main aim/objective of this study was to detect and characterize theBrucella speciesfrompatients having complaints of joint pain and also to know thepotential causes of human brucellosis. In our study, we focused on joint pain symptoms that may be due to arthralgia or arthritis. Introduction: Brucellosis is a neglected zoonotic disease that affects both humans and animals.In humans, brucellosis begins with chronic illness leading to greatfinanciallosses fromnot being able to work welland continued treatment costs, butfew such studies have come from northern India.Joint pain is the common presentation of brucellosis and there are several risk factors associated with brucellosis. A total of 200 blood samples were collected from the participants having joints pain from September 2019 to September 2021 at Gandhi Memorial & Associated Hospitals ofKing George's Medical University, Lucknow, India, and tested by serology for anti-BrucellaIgM and IgG enzyme-linked immunosorbent assay (ELISA),moleculartests byreverse transcriptase-polymerase chain reaction (RT-PCR),conventional polymerase chain reaction (PCR), and automated blood culture system.The anti-Brucella IgM and IgG ELISA were performed using the kit from NovaTec Immundiagnostica GmbH (Dietzenbach, Germany). Isolation of DNAwas carried out using the QIAamp DNA Mini kit (QIAGEN,Hilden, Germany), and the primers and probes specific for targeted regions (BCSP31 and IS711 gene)in theBrucellagenome were procured from Eurofins Scientific SE (Luxembourg, France), and for internal control from CDC. The study showed 19(9.5%) and 23 (11.5%) positive results by anti-BrucellaIgM ELISA and anti-BrucellaIgG, respectively, andof these, one (0.5%) was positive for both anti-BrucellaIgM and anti-BrucellaIgG ELISA.Out of 19 anti-Brucella IgM ELISA positive, eight (4%) samples were positive forPCR/RT-PCRandthat was negative for anti-Brucella IgG ELISA. All blood culture reports of all patients were negative. Conclusion: Anti-Brucella IgM ELISA was more accurate than anti-Brucella IgG ELISA in detecting human brucellosis. Consumption of animal products(i.e. milk, a dairy product of cow, buffalo, goat, and meat of goat)and contact with animals were themain risk factors that were identifiedfor Brucelladisease.

Short Tandem Repeat (STR) Profiling of Earwax DNA Obtained from Healthy Volunteers.

The present study aimed to establish human earwax as a potential source of DNA evidence that could be effectively used in human identification. Sixty earwax samples were obtained from 15 healthy male and female Saudi volunteers living in Riyadh, Saudi Arabia. Four consecutive earwax swab samples were obtained from each volunteer and stored for 1, 15, 30 and 60 days. Earwax samples were stored at room temperature (20-22 °C). Reference oral swab was also taken from each volunteer. DNA was extracted by QIAamp DNA Mini kit and quantified by real-time polymerase chain reaction (RT-PCR) on 7500 Thermal Cycler. Autosomal STR loci were amplified using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Amplified fragments were size separated and analyzed on a 3500 Genetic Analyzer. Complete autosomal STR profiles were obtained from the earwax swabs of all the volunteers stored up to 30 days after the collection. Some STR profiles were partially obtained 60 days after the earwax collection. Allelic drop-out, allelic drop-in, and stutters were seen in earwax samples analyzed 60 days after the collection. The results have shown that human earwax can be a potential source of DNA evidence for human identification up to 30 days after the earwax collection. It is recommended to quickly analyze earwax samples or store them at room temperature or at -10 °C after their recovery from the crime scene.

An improved rapid method for DNA recovery from cotton swabs.

We present a novel rapid method for the recovery of cellular and free DNA from cotton swabs based on a simple elution buffer containing a high molecular weight polymer and detergent combined with a short proteinase K digestion to release cellular DNA. This method shows increased yields approaching 80% recovery of the input DNA compared to the QIAamp DNA Mini kit standard extraction protocol for swabs which has a recovery of 20-30%. The buffer components in the described method are compatible with direct PCR analysis of the isolated DNA without further purification. Recovery efficiencies were estimated by qPCR.

Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used.

Whole genome sequencing data of Candida krusei, the pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia.

Candida krusei is a Candida non-albicans species with a high prevalence, which causes candidaemia. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections; however, it is only a fungistatic against Candida spp., and both inherent and acquired resistance to fluconazole have been reported. C. krusei species is also reported as the only Candida sp. which has an intrinsic resistance factor to fluconazole. Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei. The purpose of this study was to investigate the genome of clinical C. krusei isolates and correlate the resistant phenotypes with mutations in resistance genes. A total of 16 samples of C. krusei from clinical samples from hospitals in Jakarta were used in the experiment. All colonies were extracted using the QIAamp DNA Mini Kit. The library was prepared using the Illumina DNA Prep Kit. The sequencing process was carried out on the Illumina MiSeq Platform using a 2x301 paired-end configuration. FASTQ raw files are available under the BioProject Accession Number PRJNA819536 and Sequence Read Archive Accession Numbers SRR18739949 and SRR18739964.

Molecular Characteristics of Macrolide Resistance in Treponema pallidum from Patients with Latent Syphilis in Xinjiang, China.

Macrolide resistance in Treponema pallidum (T. pallidum) has been increasing in recent years worldwide. However, few data are available on macrolide resistance in T. pallidum from Xinjiang province, located in the western part of China, which is an area with a high incidence of syphilis. In this study, we investigated the molecular characteristics of macrolide resistance in T. pallidum from patients with latent syphilis in Xinjiang, China. In total, 204 whole blood samples were collected from patients with latent syphilis during 2016 to 2017, in the First Hospital of Xinjiang Medical University. Genomic DNA of blood samples was extracted using a QIAamp DNA Mini Kit and T. pallidum was detected by PCR with the specific polA gene of T. pallidum. The 23S rRNA gene of T. pallidum was amplified among the T. pallidum-positive samples by nested PCR, and macrolide resistance-associated mutation sites A2058G and A2059G in the 23S rRNA gene were identified using restriction enzymes MboII and BsaI. The specific polA gene of T. pallidum (T. pallidum positive) was detected in 27 blood samples (13.2%) from 204 patients with latent syphilis. The 23S rRNA gene was then amplified in all 27 T. pallidum-positive samples, among which 24/27 samples (88.9%) harbored the A2058G mutation in the 23S rRNA gene and 3/27 (11.1%) had the A2059G mutation. Our results indicated that T. pallidum macrolide resistance should not be ignored in Xinjiang, China, and that A2058G was the predominant macrolide resistance mechanism. Blood may be a suitable specimen for the detection of resistant mutations of T. pallidum in patients with latent syphilis who do not show any clinical symptoms.

Telomere length from peripheral blood DNA in major depressive disorder: a case/control comparison and association with antidepressant treatment response

IntroductionMajor depressive disorder (MDD)is one of the most common psychiatric disorders and a large number of patients present a poor response to treatment. Shortening of telomeres physiologically occurs after each cell division and their shortening is also associated with cell ageing. Studies are affirming that patients who suffer from mental illnesses have shorter telomeres in comparison with patients without such affections. 4 The effect of antidepressant medication on the biology of the telomeres was studied very little in humans and the current data suggest that the length of telomeres is a promising target regarding the prognosis and the response to psychotropic treatment.ObjectivesTo analyze the telomere length (TL) from peripheral blood DNA of patients with major depressive disorder (MDD) compared to healthy controls. A second objective was to compare the TL of patients in relation to antidepressant treatment.MethodsWe analysed the clinical data from 16 patients admitted to the Psychiatry Clinic of Timisoara with the diagnosis of MDD and 10 healthy controls. The collection of clinical data was carried out in a structured and standardized manner both on paper and electronically, and the Hamilton Depression Rating Scale (HDRS) was applied to assess the severity of depression. Also, blood samples were collected, and plasma and white blood cells (WBC) were separated by centrifugation. Patients’ samples were collected before and after 12 weeks of escitalopram antidepressant treatment, and a structured diagnostic interview and a standardized depression rating scale were done. DNA was extracted from WBC using the QIAamp DNA Mini Kit (Qiagen), and TL was determined by real time PCR using the Absolute Human Telomere Length Quantification qPCR Assay Kit (Sciencell Research Laboratories) according to the manufacturers’ specifications. The TL expressed as megabases/diploid cell (Mb/DC) were compared between cases and controls using a Mann-Whitney test, and between patients before and after treatment using a Wilcoxon matched-pairs signed rank statistical test.ResultsThe mean±SD telomere length for healthy controls was 0.3614±0.082Mb/DC, for treatment naïve patients was 0.4513±0.199Mb/DC, and for patients after treatment was 0.3476±0.070Mb/DC. There was no statistical significant difference in TL between patients and controls (p=0.266), nor between patients before and after treatment (p=0.055).ConclusionsIn this pilot project of limited sample size there was no difference in TL between MDD patients and healthy controls. Moreover, the TL of patients did not significantly change after 12 weeks of escitalopram antidepressant treatment.Disclosure of InterestNone Declared

Whole-genome sequencing of Histophilus somni strains isolated in Russia.

Histophilus somni is a Gram-negative bacterium belonging to the Pasteurellaceae family that can cause bovine histophilosis. Histophilus may act as a commensal or opportunistic bacterial cattle pathogen. Comparing genomes of the pathogenic strain 2336 with the non-pathogenic preputial 129Pt isolate revealed some putative virulence factors. The study of the complete genomes of H. somni strains circulating in Russia has never been conducted before. This study aimed to identify genetic features of the H. somni strains isolated in Russia and evaluate the possibility of using strains for vaccine development. Three strains of H. somni were isolated from different sources. Strain 188-VIEV was isolated from a vaginal swab sample of cattle with endometritis. 532-VIEV and 551-VIEV were cultured from the cryopreserved bull semen samples imported from Canada. Histophilus somni strain ATCC 700025 provided by ATCC (American Type Culture Collection) was also used in the study. DNA extraction was performed using QIAamp DNA Mini Kit (QIAGEN, USA). The whole-genome sequencing of the four strains was performed using Illumina Miseq. The comparison of the resulting sequences with the complete genomes of H. somni 2336 and 129Pt, and detection of the resistance genes and virulence factors, was performed using the ResFinder and Virulence Factor Database web services. The genome size of the samples varied from 1.9 to 2.3 Mb. The number of coding sequences varied from 1795 to 2256. The average sequence density was 90%. The total guanine-cytosine (GC) content was 36.8%-37.2%, which coincided with data previously obtained for H. somni. Three out of four studied strains encoded putative virulence factors such as filamentous hemagglutinin homologs, lipooligosaccharide biosynthesis proteins, and proteins involved in iron transport and utilization. The Ser83Ile substitution was identified in the DNA topoisomerase II (gyrA) in H. somni strains 532-VIEV and 551-VIEV cultured from bull semen which led to resistance to fluoroquinolones. The gene (AAC-6-Ia + APH-2'') encoding a bifunctional aminoglycoside modification enzyme was detected in strain 551-VIEV. Strains with virulence genes identified could be candidates for designing vaccines and potentially represent antigen sources. The results show that antibiotic-resistant H. somni can be spread with semen used for artificial insemination.

Interleukin-6 Gene Polymorphisms Influencing in hematological indices from sickle cell Anemia Patients

The homozygous hemoglobin SS is characterized Sickle Cell Anemia (SCA), altering the original structure of erythrocytes to a sickle shape. The hemoglobinopathies encompass all genetic diseases of hemoglobin and the SCA is the one that presents the greatest clinical manifestations variability and also the most severe ones, causing chronic hemolysis, vaso-occlusive crises and severe anemia in patients. The present study aimed to investigate the role of rs2069832, rs2069835, rs2069840, rs2069845 and rs2069849 polymorphisms in the Interleukin-6 gene in the hematological values of SCA patients treated at Fundação HEMOAM, Manaus, AM. The inclusion of patients was carried out through outpatient care at HEMOAM. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) and molecular analyzes by TaqMan® probes on Applied Biosystems QuantStudio 6 Flex Real-Time PCR System. A total of 277 SCA patients were included in this study, having the female gender having a minimally higher frequency (55.3%). The mean age at diagnosis was approximately three years old, with brown race being the most predominant (77.6%). The rs2069832_AA and rs2069845_AA genotypes showed high values for red blood cell, hemoglobin and hematocrit indices, by having an important role as a protective factor for hemolysis in these patients. While the rs2069835_CC genotype showed decreased values for the same hematimetric indices, demonstrating to be a potential risk factor for increased hemolysis. No significant correlation in hematimetric indices was observed for the rs2069840 and rs2069849 genotypes. There are few studies correlating the genetic variants of the IL-6 gene in SCA in the state of Amazonas, however, it is known that IL-6 is involved in cell proliferation and greater response to inflammatory cytokines, and may modulate the clinical response in these patients, such as chronic hemolysis, vaso-occlusion and infections. Our work demonstrated associations of risk and protective IL-6 genotypes for possible hemolysis in patients with sickle cell anemia. We understand that an investigation with a larger number of patients would be recommended to elucidate the roles of the studied polymorphisms in sickle cell anemia. In addition, elucidating the role of Il-6 in sickle cell anemia may lead to the development of new strategies and therapies to prevent the systemic effects of excessive cytokine production and, consequently, reduce the severity of crises in these patients, providing better prognosis, clinical follow-up and welfare.

Comparative Study of Two Methods of DNA Extraction from Dental Pieces

Colombia is a country with an internal armed conflict of more than 60 years, where forensic sciences claim a vital role in strengthening transitional justice within the framework of the peace process. Just look at the statistics of unidentified bodies, to realize how far behind the identification process is in Colombia; where according to Life application: There are some 26,660 to 35476 unidentified bodies that, despite the joint efforts of agencies such as the Missing Persons Search Unit, Forensic Medicine and the Attorney General’s Office, have not yet been handed over to their relatives. For this reason, it is necessary to recognize the massive task of forensic specialists to propose new methods that optimize the identification process. Responding to this call, the IdentiGEN Laboratory compared two methods of extracting DNA from teeth, the ChargeSwitch Forensic DNA Purification Kit (Invitrogen) and the QIAamp DNA Mini Kit (Qiagen) to determine which of the two allowed optimization, not only the time of analysis but the costs of application with respect to the conventional method used for these matrices. To do this, DNA extraction was performed with each kit and subsequently extracts were quantified by fluorometry, demonstrating that although both methods are highly efficient in time and cost; the QIAamp method DNA Mini Kit (Qiagen) has a higher yield in terms of the amount of genetic material obtained. Finally, the dental parts analyzed using the PowerPlex Fusion System (Promega) commercial kit were successfully typified, showing not only that these matrices present a viable source of DNA collection but that it is possible to continue improving extraction methods so that they are increasingly simple, economical and effective.

Abstract P058: Rapid, noninvasive, office-based, esophageal cell collection for early detection of esophageal precancer and cancer

Abstract Purpose: Esophageal Adenocarcinoma (EAC) is the second most lethal (<20% five-year survival) and fastest growing (>500% increase in incidence over 40 years) cancer in the U.S. All EAC is believed to arise from a metaplastic esophageal precancer, Barrett’s Esophagus (BE), in at-risk patients with gastroesophageal reflux disease (GERD). Non-dysplastic BE can be monitored for progression to dysplastic BE, which can be ablated endoscopically, reliably halting progression to EAC. Since mortality is high even in early stage EAC (<50% five-year survival), clinical practice guidelines recommend BE screening in GERD patients with multiple risk factors. Since, endoscopic BE screening has failed (<10% compliance with guidelines), recently updated guidelines from both the American College of Gastroenterology and American Gastroenterological Association now support non-endoscopic biomarker screening as an acceptable alternative to endoscopy. EsoCheck®, a noninvasive swallowable balloon capsule catheter device, is the only FDA 510(k)-cleared esophageal cell collection device which permits anatomically targeted and protected sampling of distal esophageal mucosal cells. The sample is collected in a less than five-minute office procedure, without sedation or anesthesia, and sent to a central laboratory for molecular biomarker testing. We report our initial clinical experience using EsoCheck® for collection of esophageal cells in outpatient test centers. Experimental Design: 687 patients underwent EsoCheck® distal esophageal cell sampling by a trained nurse practitioner (average sampled length 5 cm). Patient tolerance was rated on a five-point scale for gag reflex (GR) severity: 1 = no GR; 2 = minimal GR; 3 = mild GR; 4 = severe GR; and 5 = worst GR therefore collection could not be completed. DNA was extracted using the QIAamp DNA Mini kit and quantitated using Qubit™ dsDNA Assay kit. Cytologic assessment using H&E staining was performed on 15 samples. Results: 98% of patients successfully completed the procedure. 88% tolerated it well (rating ≤ 3); 10% experienced severe gagging (rating 4); and only 2% were unable to complete the procedure (rating 5). Average procedure time was 3.5 min (range: 1 min to 15 min). Average DNA yield was 868 ng (range: 0 ng to 19 µg) and 92% yielded at least 100 ng. The samples undergoing cytologic assessment averaged 500,000 cells (range: 100,000 to 2.5 million). Conclusion: Rapid, noninvasive, office-based, anatomically-targeted, and protected sampling of distal esophageal mucosal cells using EsoCheck® can be successfully performed in nearly all patients and is broadly well-tolerated. Cellular and DNA yields were very good and are expected to improve with clinical experience and assay optimization. When combined with a highly sensitive molecular biomarker test, the EsoCheck® device has the potential to significantly improve BE screening compliance and prevent EAC deaths. Citation Format: Suman M. Verma, Ivy T. Le, Abhisek B. Ghosal, Marci L. Hageman, Victoria T. Lee, Brian J. deGuzman, Lishan Aklog. Rapid, noninvasive, office-based, esophageal cell collection for early detection of esophageal precancer and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P058.

Optimization of DNA extraction and sampling methods for successful forensic microbiome analyses of the skin and saliva.

Microbiome studies have contributed to many fields, such as healthcare and medicine; however, these studies are relatively limited in forensics. Microbiome analyses can provide information, such as geolocation and ancestry information, when short tandem repeat (STR) profiling fails. In this study, methods for DNA extraction and sampling from the skin and saliva were optimized for the construction of a Korean Forensic Microbiome Database (KFMD). DNA yields were estimated using four DNA extraction kits, including two automated kits (Maxwell® FSC DNA IQ™ Casework Kit and PrepFiler™ Forensic DNA Extraction Kit, updated) and two manual kits (QIAamp DNA Mini Kit and QIAamp DNA Micro Kit) commonly used in forensic DNA profiling laboratories. Next-generation sequencing of the 16S rRNA V4 region was performed to analyze microbial communities in samples. The Bacterial Transport Swab with Liquid Media (NobleBio), two cotton swabs (PoongSung and Puritan), and nylon-flocked swabs (NobleBio and COPAN) were tested for DNA recovery. The PrepFiler and Maxwell kits showed the highest yields of 3.884ng/μL and 23.767ng/μL from the scalp and saliva, respectively. With respect to DNA recovery, nylon-flocked swabs performed better than cotton swabs. The relative abundances of taxa sorted by DNA extraction kits were similar contributions; however, with significant differences in community composition between scalp and saliva samples. Lawsonella and Veillonella were the most abundant genera in the two sample types. Thus, the Maxwell® FSC DNA IQ™ Casework Kit and nylon-flocked swab (NobleBio) were optimal for DNA extraction and collection in microbiome analyses.

Direct Detection of Pseudomonas aeruginosa from Respiratory Samples of Children with Cystic Fibrosis

Introduction: Pseudomonas aeruginosa is the most important cause of lung infection and major cause of morbidity and mortality among Cystic Fibrosis patients. Early detection of P. aeruginosa from clinical samples is a key element in patient management. Appropriate antimicrobial treatment will postpone the transition to chronic lung infection.
 Methods: Sputum and deep pharyngeal swab samples (DPS) of CF patients were inoculated into conventional agar plates and identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. DNA was extracted with a column based QIAamp DNA mini kit from the samples. Molecular detection was done by using 23S rDNA specific primers with Taqman probe for quantitative PCR (qPCR) and exoA gene specific primers for in house PCR.
 Results: A total number of 67 sputum and 33 DPS samples were included to the study. Median age of the patients was 6 (2-14) for DPS and 15(7-33) for sputum samples. Detection limit was 103 cfu/ml for qPCR which displayed 94% sensitivity and 93% specificity for sputum; 44% sensitivity and 100% specificity for DPS. In house PCR displayed 94% sensitivity and 93% specificity for sputum; 50% sensitivity and 100% specificity for DPS. Time reqired for conventional method was 48 hours, 6 hours for in- house PCR and 4 hours for qPCR.
 Conclusion: Although culture is a reliable detection technique, a more rapid and sensitive way to detect P. aeruginosa from CF airway samples is essential. In our study which culture is accepted as a gold standard; PCR results is promising especially for sputum samples. Validation of molecular methods is necessary before implementation in routine laboratories. Clinical implications of discrepant results between culture and PCR detection should be carefully evaluated and combining both approaches considering antibiotic treatment could be reasonable.

Comparison of DNA Extraction Methods for the Quantification of Listeria monocytogenes in Dairy Products by Real-Time Quantitative PCR>

Listeria monocytogenes is a common foodborne pathogen affecting public health. Thus, detecting L. monocytogenes, even at low levels, in food matrices is essential. However, the current culture methods used for its detection and quantification are time consuming and difficult owing to background flora and interference by food matrices. DNA-based assays depend on DNA extraction and purification techniques. No optimal DNA extraction kit has been developed for analyzing L. monocytogenes in dairy products by real-time quantitative PCR (RT-qPCR). Therefore, in this study, we aimed to determine the efficiency of three DNA extraction kits for detecting L. monocytogenes in dairy products by RT-qPCR. We tested the efficiency of three commercial kits for DNA extraction from L. monocytogenes artificially inoculated in milk and dairy products. For the PrepSEQ rapid spin sample preparation kit and Exgene Cell SV mini, the limit of detection of was 100, 100, and 101 CFU/mL L. monocytogenes in milk, processed cheese, and infant formula, respectively, whereas that of the QIAamp DNA mini kit was 101, 103, and 102 CFU/mL, respectively. In addition, the Exgene Cell SV mini was better than the PrepSEQ rapid spin sample preparation kit for obtaining a standard curve for RT-qPCR of L. monocytogenes DNA in milk and dairy products, with a high correlation coefficient and amplification efficiency. The results of this study may be valuable for diagnostic laboratories and for developing an effective extraction method for processing food samples, such as dairy products, to subsequently detect and quantify L. monocytogenes by RT-qPCR.

Targeted Sequencing of Germline Breast Cancer Susceptibility Genes for Discovering Pathogenic/Likely Pathogenic Variants in the Jakarta Population.

Germline predisposition plays an important role in breast cancer. Different ethnic populations need respective studies on cancer risks pertinent to germline variants. We aimed to discover the pathogenic and likely pathogenic variants (P/LP-Vs) of germline breast cancer susceptibility genes and to evaluate their correlation with the clinical characteristics in Jakarta populations. The pure DNA was extracted from the blood buffy coat, using reagents from the QIAamp DNA Mini Kit® (Qiagen, Hilden, Germany). The DNA libraries were prepared using the TargetRich™ Hereditary Cancer Panel (Kailos Genetics®, Huntsville, AL, USA). The barcoded DNA libraries were sequenced using the Illumina NextSeq 500 platform. In-house bioinformatics pipelines were used to analyze the gene variants. We identified 35 pathogenic and likely pathogenic (P/LP-Vs) variants (28 frameshift, 5 nonsense, and 2 splice-site variants). The P/LP-Vs group was statistically significantly different in luminal B status (p < 0.05) compared with the non-P/LP-Vs group. The P/LP-Vs found both in BRCA1/2 genes and non-BRCA genes may increase the risk of breast cancer and alter drug responses. The screening of multigene variants is suggested, rather than BRCA testing only. Prior knowledge of the germline variants status is important for optimal breast cancer diagnosis and optimal therapy.

P-026 Lack of association between single polymorphic variants of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase 3, and 4L (MT-ND3 and MT-ND4L) and male infertility

Abstract Study question Is there any association between male infertility and the polymorphic variants of Mitochondrial Nicotinamide Adenine Dinucleotide Hydride dehydrogenase (NADH) Subunit 3 and 4L (MT-ND3 and MT-ND4L)? Summary answer The genotypes and allele frequencies of the study population have shown a lack of statistically significant association between MT-ND3 and MT-ND4L SNPs and male infertility. What is known already Male infertility is a multifactorial condition associated with different genetic abnormalities in at least 15%–30% of cases. The rate of mutations in the mtDNA is high due to the lack of histones and DNA repair mechanisms. Moreover, mtDNA is more vulnerable compared to nuclear DNA due to the lack of an efficient repairing system and high exposure to oxidative species produced by the mitochondria. Eventually, this leads to abnormal sperm function, structure and even infertility. Study design, size, duration Prospective study was carried out between 2018 and 2019. 112 semen samples were collected in this study. Participants/materials, setting, methods The present study was carried out at the department of Obstetrics and Gynecology, University of Saarland,Germany. After semen analysis, samples were purified. Mitochondrial DNA was extracted using a QIAamp DNA Mini Kit, then, the mitochondrial DNA was amplified by using the REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify MT-ND3 and MT-ND4L genes. After that samples were purified and sequenced using the Sanger method in the Microsynth Seq Lab in Germany. Main results and the role of chance Eight single nucleotide polymorphisms (SNPs) in MT-ND3 (rs2853826, rs28435660, rs193302927, rs28358278, rs41467651, rs3899188, rs28358277 and rs28673954) and seven SNPs in MT-ND4L (rs28358280, rs28358281, rs28358279, rs2853487, rs2853488, rs193302933 and rs28532881) were detected and genotyped. The genotypes and allele frequencies of the study population have shown a lack of statistically significant association between MT-ND3 and MT-ND4L SNPs and male infertility. Besides, no statistically significant difference was found between the asthenozoospermia, oligozoospermia, teratozoospermia, asthenoteratozoospermia, oligoasthenoteratozoospermia and oligoteratozoospermia subgroups of subfertile males. However, rs28358278 genotype of the MT-ND3 gene was reported in the subfertile group but not in the fertile group, which implies a possible role of this SNP in male infertility. Limitations, reasons for caution The size number of the study samples. Wider implications of the findings Further studies are required to evaluate these findings. Moreover, the subfertile individuals who exhibit a polymorphism at rs28358278 require further monitoring and evaluation. Trial registration number Basic Science

First exploratory study of bacterial pathogens and endosymbionts in head lice from a Mayan community in southern Mexico

Lice represent one of the most neglected group of vectors worldwide, particularly in Latin America. Records of bacterial agents related to head lice are non-existent in this region of the continent. Many of these communities often do not have adequate access to public services and/or health protection. The normalization of this condition prevents them from manifesting discomfort, such as bites and itching, which further aggravates the situation, as they can be vectors of important diseases. For this reason, the aim of this work was to identify the richness of bacterial pathogens (Acinetobacter, Bartonella, and Rickettsia) and endosymbionts (Wolbachia) in head lice of paediatric patients from the indigenous municipality of Hoctun, Yucatan, Mexico. DNA extraction was performed using the QIAamp DNA Mini Kit. For the detection of bacterial pathogens, fragments of the gltA, rpoB, and 16S rDNA genes were amplified. For the detection of Wolbachia, the wsp gene was amplified. Of the 28 lice analysed, the presence of two genera of bacterial pathogens was detected Acinetobacter (42.9%=12/28) and Bartonella (7.14%=2/28). We also detected the endosymbiont Wolbachia (71.42%=20/28). Our results showed that DNA from three bacteria species (Acinetobacter baumannii, Bartonella quintana, and Wolbachia pipientis) was present with frequencies ranging from 3.57% to 71.42%. This work represents the first exploratory study of the diversity of agents associated with head lice (Pediculus humanus capitis) in Mexico and Latin America. Due to the findings generated in the present study, it is important to perform surveillance of head lice populations to identify the degree of spread of these pathogens and their impact on populations in the region.

PD-4 Genetic aberration from normal tissues adjacent to biliary tract cancers

Chronic inflammation has emerged as a main mediator of the adenoma-carcinoma sequence in the carcinogenesis of biliary tract cancers (BTCs). Therefore, normal tissues adjacent to the tumor (NATs) may harvor tumor-related genetic abberations due to chornic inflammation such as sclerosing cholangitis. The purpose of this study was to determine the differences of genetic alterations between BTCs and NATs in surgically resected specimen. We describe successful NGS-based testing of 13 paired peripheral blood, NATs and BTCs from patients with 4 distal cholangiocarcinomas, 2 hilar cholangiocarcinomas, 3 intra- hepatic cholangiocarcinomas and 4 gallbladder cancers. Tissues from biliary tract cancers and normal tissues adjacent to the tumor were frozen in liquid nitrogen < 30 min after surgery and stored at −80°C until the time of DNA extraction. DNA was purified using the QIAamp DNA Mini Kit (Qiagen). Whole genome and exome sequencing was performed using the illumina HiSeq 2000 sequencing platform. Genetic aberrations were more frequently founded in NATs than BTCs. Three hyper-mutated BTC samples shared more than 3000 genetic mutations (more than 90% of total genetic mutations) with NATs. But in cases of non-hypermutated tumor, only 23% of mutations were shared between BTC and NAT. NCOR2, MUC16, CHD4, PDE4DIP, ZFHX3, TP53, ARID1B, SMAD4, ELF3, RECQL4 and RBM10 were common BTCs-related genes. ANKRD36, IQCA1, PDE4DIP, COL6A6, TP53 and ARID1B appeared in both BTCs and NATs. ANKRD36:chr2:97163323: p.Arg301Thr was the most common mutation in NATs, but the correlation with ANKRD36 and BTCs are unknown until now. We revealed several mutations may be valuable for detecting an early carcinogenesis, but further studies are needed.