QIAamp DNA Mini Kit Research Articles

Whole genome sequencing data of Candida krusei, 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 Detection and Glycoprotein B (UL55) Genotyping of Cytomegalovirus among Sudanese Renal Transplant Recipients.

Background Cytomegalovirus (CMV) is the most common opportunistic pathogen among renal transplants with significant morbidity and mortality. This study was designed to detect CMV DNA and to determine the frequency of different glycoprotein B (UL55) genotypes among Sudanese renal transplant recipients. Methods One hundred and four renal transplant recipients were included in this study. A blood specimen was collected from each recipient. DNA was extracted from plasma using the QIAamp DNA mini kit. CMV amplification and quantification were performed using CMV Real-RT Quant kits. Genotyping of human CMV gB was carried out by nested PCR and sequencing of the highly diverse region of gB. Results CMV DNA was detected in 40/104 (38.5%) of renal transplant recipients. The average of the CMV DNA viral load was 358 × 104 copies/ml (6.5 log10) ranging from 62 copies/ml (1.8 log10) to 1.43 × 108 copies/ml (9 log10). CMV viremia was detected in 60% of recipients of less than 1–12 months, 17% of 13–24, 10% of 25–36, 5% of 37–48, and 8% in more than 48 months posttransplantation with no association (p = 0.296) between CMV viremia and postrenal transplantation time. The association between the type of immunosuppressive drugs and high viral loads (>1000 copies/ml) showed a significant difference (p = 0.05). The association between CMV loads of >1000 copies/ml and symptoms of CMV disease was highly significant (p ≤ 0.001). Fever 7 (41%), fever and leucopenia 6 (35%), and gastrointestinal disease 4 (24%) were the most common symptoms of CMV disease. CMV genotyping revealed 8 cases (80%) for gB3 and 2 cases (20%) for gB4 genotypes. The most frequent genotype among Sudanese renal transplant recipients was gB3. Conclusions The frequency of CMV DNA is high among Sudanese renal transplant recipients. CMV gB3 is the most predominant glycoprotein B genotype in Sudanese renal transplant recipients.

The lung microbiome in HIV-positive patients with active pulmonary tuberculosis

Tuberculosis poses one of the greatest infectious disease threats of our time, especially when associated with human immunodeficiency virus (HIV) infection. Very little data is available on the lung microbiome in pulmonary tuberculosis (PTB) in HIV-positive patients. Three patient cohorts were studied: (i) HIV-positive with no respiratory disease (control cohort), (ii) HIV-positive with pneumonia and (iii) HIV-positive with PTB. Sputum specimens were collected in all patients and where possible a paired BALF was collected. DNA extraction was performed using the QIAamp DNA mini kit (QIAGEN, Germany) and extracted DNA specimens were sent to Inqaba Biotechnical Industries (Pty) Ltd for 16S rRNA gene sequence analysis using the Illumina platform (Illumina Inc, USA). Data analysis was performed using QIMME II and R Studio version 3.6.2 (2020). The lung microbiomes of patients with PTB, in the context of HIV co-infection, were dominated by Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Loss of biodiversity and dysbiosis was found in these patients when compared to the HIV-positive control cohort. Microbial community structure was also distinct from the control cohort, with the dominance of genera such as Achromobacter, Mycobacterium, Acinetobacter, Stenotrophomonas and Pseudomonas in those patients with PTB. This is the first study to describe the lung microbiome in patients with HIV and PTB co-infection and to compare findings with an HIV-positive control cohort. The lung microbiomes of patients with HIV and PTB were distinct from the HIV-positive control cohort without PTB, with an associated loss of microbial diversity.

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda.

In Uganda, Artemether-Lumefantrine and Artesunate are recommended for uncomplicated and severe malaria respectively, but are currently threatened by parasite resistance. Genetic and epigenetic factors play a role in predisposing Plasmodium falciparum parasites to acquiring Pfkelch13 (K13) mutations associated with delayed artemisinin parasite clearance as reported in Southeast Asia. In this study, we report on the prevalence of mutations in the K13, pfmdr-2 (P. falciparum multidrug resistance protein 2), fd (ferredoxin), pfcrt (P. falciparum chloroquine resistance transporter), and arps10 (apicoplast ribosomal protein S10) genes in Plasmodium falciparum parasites prior to (2005) and after (2013) introduction of artemisinin combination therapies for malaria treatment in Uganda. A total of 200 P. falciparum parasite DNA samples were screened. Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) procedure. The PCR products were sequenced using Sanger dideoxy sequencing method. Of the 200 P. falciparum DNA samples screened, sequencing for mutations in K13, pfmdr-2, fd, pfcrt, arps10 genes was successful in 142, 186, 141, 128 and 74 samples respectively. Overall, we detected six (4.2%, 6/142; 95%CI: 1.4-7.0) K13 single nucleotide polymorphisms (SNPs), of which 3.9% (2/51), 4.4% (4/91) occurred in 2005 and 2013 samples respectively. All four K13 SNPs in 2013 samples were non-synonymous (A578S, E596V, S600C and E643K) while of the two SNPs in 2005 samples, one (Y588N) is non-synonymous and the other (I587I) is synonymous. There was no statistically significant difference in the prevalence of K13 (p = 0.112) SNPs in the samples collected in 2005 and 2013. The overall prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186, 95%CI: 25.1-50.4). Of this, 4.2% (4/95), 76.9% (70/91) occurred in 2005 and 2013 samples respectively. In 2005 samples only one SNP, Y423F (4.2%, 4/95) was found while in 2013, Y423F (38.5%, 35/91) and I492V (38.5%, 35/91) SNPs in the pfmdr-2 gene were found. There was a statistically significant difference in the prevalence of pfmdr-2 SNPs in the samples collected in 2005 and 2013 (p<0.001). The overall prevalence of arps10 mutations was 2.7% (2/72, 95%CI: 0.3-4.2). Two mutations, V127M (4.5%: 1/22) and D128H (4.5%: 1/22) in the arps10 gene were each found in P. falciparum parasite samples collected in 2013. There was no statistically significant difference in the prevalence of arps10 SNPs in the samples collected in 2005 and 2013 (p = 0.238). There were more pfmdr-2 SNPs in P. falciparum parasites collected after introduction of Artemisinin combination therapies in malaria treatment. This is an indicator of the need for continuous surveillance to monitor emergence of molecular markers of artemisinin resistance and its potential drivers in malaria affected regions globally.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Variation in White Blood Cell Differential Fluorescence Scattergram: A Clue for Automated Malaria Diagnosis

Background: Malaria is an arthropod-borne disease caused by Plasmodium. Malaria is a significant public health challenge with about 3.3 billion human lives at risk in 97 countries with two hundred million estimated malaria cases and 600,000 fatalities. Timely diagnosis can prevent disease spread, loss of human lives, and its harmful impact on the economy therefore malaria diagnosis by automated hematology analyzer is of great value. Objective: To assess identification of scattergram abnormalities in malaria samples and to compare its performance with Microscopy, RDTs &amp; PCR. Study Design: Descriptive and cross-sectional study Place and Duration of Study: Department of Pathology, Liaquat University of Medical &amp; Health Sciences Jamshoro, 06 months from May 2020 to October 2020. Methodology: Eighty seven malaria suspected samples were included. CBC performed on Sysmex XN1000i followed by Microscopy of thin blood film, RDTs by using STANDARDTM Q Malaria P.f/Pan Ag Test, DNA extracted by QIAamp DNA mini kit, PCR performed by GenoAmp Real-time qPCR Malaria. Results: The median age of patients was 24+13.93 years. WDF Scattergram abnormalities were observed in 95.4 %of samples with sensitivity of 97.6% and specificity of 100% respectively. PPV was 100% and NPV was 99.8%. 96.4% of patients were malaria positive by microscopy. ICT was positive in 96.6% of samples while PCR confirmed plasmodium presence in 97.7% samples. Conclusion: Automated hematology analyzer provides high sensitivity and specificity for malaria diagnosis. The Sysmex XN-1000i can detect certain abnormalities in WDF scattergrams in malaria patients. As a result, the appearance of an abnormal WDF scattergram in a patient with a history of febrile illness, aids the pathologist in confirming a malaria diagnosis. Keywords: Malaria, Plasmodium Vivax, Plasmodium Falciparum, WDF scattergram, ICT, RT qPCR

Genetic assessment of the Forest Dormouse Dryomys nitedula (Pallas, 1773) in Latvia

The Latvian population of the Forest Dormouse is small and isolated, situated far from the main distribution range of this species. The population is located in the south-eastern region of Latvia, close to the Belarusian border. Sixty-three nest-boxes were located throughout this distribution area and captured forest dormouse individuals were sampled for genetic analysis. Animal age, sex, and coordinates from place of capture were recorded. DNA was extracted from 120 individuals using the QIAamp DNA Mini Kit (Qiagen, Germany). RAPD-PCR polymorphisms at 65 loci were used to examine the genetic diversity and differentiation of the Forest Dormouse population in Latvia. 89% of loci were polymorphic, average expected heterozygosity over all loci was 0.321, average polymorphism information content (PIC) was 0.246. The program ‘Structure’ was used to perform Bayesian assignment of individuals to a predefined number of clusters. The deltaK method identified that the most likely number of clusters within the Latvian Forest Dormouse population was three. Assignment of individuals to a particular cluster mostly corresponded with the location where the individual was captured. Analysis of molecular variance (AMOVA) indicated that there was significant genetic differentiation between the three clusters with pairwise Phipt (a modification of Fst) values ranging from 0.118 to 0.180.

Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples.

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.

BsmI gene polymorphism of vitamin D receptor in obese Egyptian male medical students and its relationship with vitamin D deficiency

BackgroundVitamin D regulates cell proliferation and differentiation and exhibits immunoregulatory, antiangiogenic, and antioxidant characteristics. Vitamin D deficiency and vitamin D receptor (VDR) gene polymorphisms have been associated with an increased risk of metabolic syndrome components such as abdominal obesity, with both vitamin D deficiency and VDR gene polymorphisms contributing to its development. Obesity, metabolic disorders, and bone mass defects are associated with VDR alleles. The importance of VDR in the etiology of obesity has been related to the existence of the VDR polymorphisms FokI, BsmI, ApaI, and TaqI. VDR expression in adipocytes affects energy metabolism and aids in obesity development.ObjectiveThe aim of this study was to examine a possible association between BsmI (B, b allele) polymorphism in the VDR gene and vitamin D status in obese Egyptian male medical students.Patients and methodsThis study was based on a comprehensive experiment known as “Nutritional Assessment of Medical Educational Students (NAMES)” that was completed in 2018 with 700 healthy participants (men and women). In total, out of the 700 participants 30 healthy men were enrolled in this study who matched our inclusion criteria. They attended Ain Shams University Hospital in Cairo between April 2019 and November 2020. This study was conducted at the Clinical Pathology Department of Ain Shams University Hospital, which is a part of the Faculty of Medicine. Participants were divided into two groups: Group I consisted of 12 obese men with nondeficient vitamin D levels (> 20 ng/dl), and Group II consisted of 18 obese men with deficient vitamin D levels (20 ng/dl). Peripheral blood samples were collected into EDTA tubes from all groups, and DNA was extracted and purified using spin purification for PCR (QIA amp DNA Mini Kit). A commercial real-time polymerase chain reaction (PCR) kit with predesigned TaqMan probes was used to analyze the single nucleotide polymorphism (SNP) (BsmI) rs 1544410 in the VDR gene.ResultsNo statistically significant relationship was observed between vitamin D levels and all InBody bioelectrical impedance characteristics, as well as BsmI gene polymorphism and all InBody bioelectrical impedance parameters (P > 0.05). Regarding Bb and BB genotypes, no statistically significant difference was observed between Groups I and II. The prevalence of BB genotype was higher in vitamin-D-deficient individuals, and Bb genotype was more common among obese participants than BB genotype, which showed a higher prevalence of the “b” gene; however, these were not significant. Iron profile (iron level, ferritin level, TIBC, and transferrin saturation) and BsmI gene polymorphism showed no statistically significant relationship (P > 0.05). Glycated hemoglobin (HbA1c) level and BsmI gene polymorphism showed a statistically significant relationship (P = 0.002), with a higher mean value among Bb genotype carriers than among BB genotype carriers. Eosinophil count showed a statistically significant difference between BB and Bb genotype carriers (P = 0.045), with a higher mean value among Bb genotype carriers than among BB genotype carriers. Bsml gene polymorphism showed no statistically significant relationship with any other complete blood count parameters (P > 0.05.)ConclusionThere was no significant relationship between VDR gene polymorphism (BsmI) and body mass index (BMI) nor between the different InBody bioelectrical impedance parameters. We noticed the prevalence of BB genotype among vitamin-D-deficient obese students and the frequency of the “b” allele among obese candidates according to the findings of our study. There was also no significant relationship between BsmI gene polymorphism and vitamin D levels. However, BsmI gene polymorphism and HbA1c levels and eosinophil count showed a relationship, which requires further investigation.

Abstract P2-11-17: Feasibility of microbiome analysis from random periareolar fine needle aspiration in premenopausal women at increased risk for breast cancer

Abstract Background: Nearly 10% of breast cancers (BC) are diagnosed in premenopausal woman under age 45 and of childbearing potential. Women considering future childbearing are typically excluded from BC prevention trials and are ineligible for standard of care chemoprevention. More biomarkers are needed to support BC prevention trials in this young cohort. Women of childbearing potential are encouraged to supplement diet with minimum of 300mg of EPA + DHA omega-3 fatty acids (FA) per day. EPA and DHA are thought to have a favorable effect on the gut microbiome implicated in cancer development. Few studies have characterized the breast microbiome by core biopsy or surgical sample but to our knowledge no studies have explored the feasibility of breast microbiome collection using the less invasive technique Random Periareolar Fine Needle Aspiration (RPFNA). In a pilot study, we demonstrated feasibility of recruiting premenopausal women considering future pregnancy to a BC prevention trial with a 6-month intervention of omega-3 FA supplementation (19-A-1921-SABCS). RPFNA was used to collect breast tissue for biomarker analysis, which is a mildly invasive technique used for repeated sample collection in BC prevention trials. Objectives: 1) To determine the feasibility of characterizing breast microbiome from specimens collected by RPFNA in premenopausal woman at high risk for BC, 2) To identify changes in the breast and stool microbiome with omega-3 FA supplementation in this population. Methods: Ten women between the ages of 21 and 40 who were considering future pregnancy and at high risk for BC were enrolled to a pilot study and took Omega-3-Acid Ethyl Ester (total of 750mg DHA and 930mg EPA) daily for 6 months. Tissue collection with RPFNA of breast as well as blood, urine and stool were completed at baseline and off-study visit. RPFNA samples from the first 2 passes at each site of breast (4 sites total) were collected for microbiome and placed in a 2mL tube with 0.5 – 1cc of PBS and flash frozen and stored at -80C. DNA was isolated from RPFNA samples using QIAamp DNA Mini Kit (51304). Microbiome profiling analysis was performed by Veracet using 16S V4 rRNA gene sequencing on the Illumina MiSeq platform. Wilcoxon signed rank test was used to compare paired samples. Results: Of the 10 women enrolled, median age was 33 years (range 22-37). 90% (9 of 10) returned for off-study visit. Of the 9 women who completed the off-study visit, 2 elected to not undergo off-study RPFNA. There were 16 total stool samples and 17 total RPFNA samples for microbiome evaluation. There were 6 paired (baseline and off-study) stool and 7 paired RPFNA samples. Mean DNA concentration from RPFNA samples was 10.36ng/µl (range 0.62 – 74.10). From all samples, 52.1% of operational taxonomic units (OTUs) were classified at the genus level. Breast samples were sequenced to a depth of mean 27,767 reads (range 5,745 – 125,445) and stool to a depth of mean 119,296 reads (range 72,979 – 188,867). The alpha diversity metric of OTU richness was 1069 (breast) and 438 (stool). Shannon diversity was 4.51 (breast) and 3.89 (stool). Mean OTU richness for baseline and off-study RPFNA samples were 1098 and 1028 respectively (V = 25, p value = 0.076). Mean OTU richness for baseline and off-study stool samples were 440 and 437 respectively (V = 15, p value = 0.40). Conclusion: We demonstrated feasibility of analyzing breast microbiome from an RPFNA specimen. Additional investigation with modifications to technique and/or sample population is. needed to achieve adequate sequencing depth for characterization of breast microbiome. Lower depth of sequencing in breast samples is thought to reflect differences in microbial DNA quantity. We were unable to assess change in microbiome composition in breast or stool samples with omega-3 fatty acid supplementation due to small sample size. Citation Format: Lauren E Nye, Jennifer R Klemp, Kandy R Powers, Anne P O'Dea, Amy L Kreutzjans, Trina Metheny, Teresa A Phillips, Susan E Carlson, Bruce F Kimler, Carol J Fabian. Feasibility of microbiome analysis from random periareolar fine needle aspiration in premenopausal women at increased risk for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-17.

Association between the single nucleotide variants of the mitochondrial cytochrome B gene (MT-CYB) and the male infertility

BackgroundIdiopathic male infertility can be attributed to genetic predispositions that affect sperm performance and function. Genetic alterations in the mitochondrial DNA (mtDNA) have been linked to certain types of male infertility and abnormal sperm function. Mutations in the mitochondrial cytochrome B (MT-CYB) gene might lead to some deficiencies in mitochondrial function. Thus, in the current study, we aimed to investigate the effect of mutations in the MT-CYB gene on sperm motility and male infertility.Methods and resultsSemen specimens were collected from 111 men where 67 men were subfertile and 44 were fertile. QIAamp DNA Mini Kit and REPLI-g Mitochondrial DNA Kit from QIAGEN were used to isolate and amplify the mitochondrial DNA. Followed by PCR and Sanger sequencing for the target sequence in the MT-CYP gene. Sequencing of the MT-CYB gene revealed a total of thirteen single nucleotide polymorphisms (SNPs). Eight SNPs were non-synonymous variant (missense variant) including: rs2853508, rs28357685, rs41518645, rs2853507, rs28357376, rs35070048, rs2853506, and rs28660155. While five SNPs were Synonymous variant: rs527236194, rs28357373, rs28357369, rs41504845, and rs2854124. Among these SNPs, three variants showed a significant difference in the frequency of the genotypes between subfertile and fertile groups: rs527236194 (T15784C) (P = 0.0005), rs28357373 (T15629C) (P = 0.0439), and rs41504845 (C15833T) (P = 0.0038). Moreover, two SNPs showed a significant association between allelic frequencies of rs527236194 (T15784C) (P = 0.0014) and rs41504845 (C15833T) (P = 0.0147) and male subfertility.ConclusionThe current study showed a significant association between the MT-CYB gene polymorphisms and the development of male infertility. In particular, rs527236194, rs28357373 and rs41504845 variants were found to be the most related to the subfertility group. Further studies on larger and other populations are required to reveal the exact role of this gene in the development of male infertility. In addition, functional studies will be helpful to elucidate the molecular impact of the MT-CYP polymorphisms on mitochondrial function.

Assessment of human parvovirus B19 infection in Egyptian hemodialysis patients.

Parvovirus B19V has been shown to be associated with end-stage renal disease (ESRD) with increased risk of post-infection anemia, especially in hemodialysis (HD) patients. This effect may be due to immunosuppression, insufficient erythropoietin, or short lifespan of red blood cells. Therefore, parvovirus infection should be investigated in this group of patients suffering from anemia or pancytopenia. We assessed the frequency of parvovirus B19 in HD patients attending Suez Canal University Hospital and analyzed the correlation of this infection with hematological parameters in those patients compared with normal individuals. We recruited 80 ESRD patients on hemodialysis and 70 healthy controls. History-taking, full examination, and complete blood count (CBC) were performed for all study subjects. Parvovirus B19 detection was performed through polymerase chain reaction (PCR), which included the QIAamp DNA Mini Kit for extracting DNA, which was amplified using TaqMan Universal Master Mix and detected using TaqMan MGB probes by real-time PCR using Rotor Gene Analyzer (6000). HD patients had a significantly higher frequency of B19V infection than the control group (p= .02). We also found that parvovirus B19-infected HD patients had significantly lower CBC values than uninfected patients. The frequency of parvovirus B19 was significantly higher in HD patients and was associated with lower hematological parameters than in uninfected patients, suggesting a significant role of this virus in the pathogenesis of anemia and/or pancytopenia in ESRD.

EmbB Gene association with Ethambutol drug Resistance in Mycobacterium Tuberculosis Patients

Background: Tuberculosis (TB) is fatal and life threatening infectious disease. The transmission rate of tuberculosis is very high. Various drugs are used as treatment for TB. Recently it has been observed that one of the most important factor for fast TB spread is development of anti-TB drug resistant mycobacterium tuberculosis (MTB). Various combination of drugs like isoniazid (INH), rifampicin (RIF), Streptomycin(SM), pyrazinamide (PZA) or ethambutol (EMB) are in global use for TB treatment. Improper usage of these drugs makes the person prone to develop anti-TB drug resistant tuberculosis. Aim: To evaluate association of embB gene with ethambutol resistance in Mycobacterium Tuberculosis. Methods: 104 Specimens of sputum from suspected tuberculosis patients were processed for inoculation in Lowenstein J Medium after it has been decontaminated properly. Kit method by using QIAamp DNA Mini kit was utilized for extraction of DNA. Then region from base 6953 to 10249 of embB gene was amplified through PCR and then followed by sequencing with the aid of softwares blast2seq and ClustalW2. Three primer sets were utilized to amplify embB gene. Ethambutol (EMB) Resistant MTB specimens were processed to study mutation in embB gene. Results: Out of the total 104 sputum specimens, 14 samples were found to have ethambutol resistance. These 14 samples were then processed for mutational analysis. DNA sequence analysis of these 14 samples confirmed embB gene mutation in 10 samples. Mutational analysis revealed that 08 samples showed mutation at codon 306 and two samples showed mutation at 319 codon. The reported mutation Methionine →Isoleucine was seen in 07 samples with ATG codon replaced by ATA codon at codon position 306. One sample showed mutation as Methionine →Isoleucine with ATG codon replaced by ATC codon at codon position 306. Two samples showed mutation as Tyrosine →Serine with TAT codon replaced by TCT at 319 codon position in embB gene. Conclusion: This study concludes that mutation of certain genes particularly point mutation of embB gene at codon 306 and 319 is associated with drug resistance of ethambutol in ethambutol resistant mycobacterium tuberculosis patients. Keywords: Ethambutol, embB gene, Mycobacterium tuberculosis.

Circulating Cell-Free DNA (ccfDNA) Isolation from Patients with Diffuse Large B-Cell Lymphoma (DLBCL): Comparative Analysis of Commercial Kits

Introduction: Isolation of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) are useful methodologies for studies of neoplastic genetic material and can provide information about cancer heterogeneity, prognosis and minimal residual disease (MRD) [1,2]. Therefore, the recovery of ccfDNA in sufficient quantity and adequate purity is crucial to provide accurate and reproducible results. In this study, we investigate the efficiency of four different commercial kits to recover short fragments of DNA compatible with the ccfDNA.Methods: Ten mL of total peripheral blood from seven patients with Diffuse Large B-Cell Lymphoma (DLBCL) were collected in EDTA and were immediately processed. Plasma was obtained by centrifugation at 1.900 x g for 10 min at 4 ºC, followed by other centrifugation at 20.000 x g for 10 min at 4 ºC to remove cellular debris. Aliquots of 1.0 mL of plasma were stored at -80 ºC until ccfDNA extraction. The ccfDNA extraction was performed using the same samples by the following kits: QIAamp MinElute ccfDNA kit (CFDNA - Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (QBLOOD - Qiagen, Hilden, Germany), Illustra Blood GenomicPrep Mini Spin Kit (GE - GE Healthcare Bio-Sciences Corp, UK) and ccfDNA isolation kit Quick-ccfDNA Serum & Plasma Kit (ZYMO - Zymo Research, Irvine, USA) [Table 1] according to the manufacturer's recommendations. The ccfDNA obtained was eluted in 30 µL of elution buffer for the subsequent experiments. The measurement of the ccfDNA obtained for each kit was performed in a Qubit 2.0 fluorometer using Qubit dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, USA). The analysis of integrity, purity and the size of the fragments of DNA were performed in the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) equipment using the High Sensitivity DNA kit (Agilent Technologies, Germany).Results: The results obtained for each commercial kit in the Qubit-analysis were: CFDNA (0.27 ng/uL), QBLOOD (0.26 ng/uL), ZYMO (0.03 ng/uL) and GE (0.02 ng/uL) [Figure 1]. The Bionalyzer eletrophoresis showed that CFDNA kit was able to recover two different fragments sizes of ccfDNA one of 360-380 bp (0.114 ng/uL) and other of 160-180 bp (1.808 ng/uL). The DNA recovered with QBLOOD revealed sizes of 360-380 bp (0.031 ng/uL) and 160-180 bp (0.079 ng/uL). Both kits, GE and ZYMO were not able to isolate fragments of DNA smaller than 10.380 pb .Conclusion: We demonstrated that QIAamp MinElute ccfDNA kit (CFDNA) was capable to recovery high quantity of small fragments of DNA compatible with ccfDNA. This result supports the use of protocols based in magnetic beads in experiments working with ccfDNA. Additionally, we highlight the importance to verify the quality and not only the quantity of the DNA extracted to confirm the presence of ccfDNA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ANALYSIS OF CYCLOPHOSPHAMIDE-INDUCED N5-NITROGEN MUSTARD FORMAMIDOPYRIMIDINE ADDUCT IN DRIED BLOOD SPOT USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Cyclophosphamide is one of the nitrogen mustard agents that is frequently used in cancer chemotherapy. Nevertheless, long-term use of high dosage cyclophosphamide has been proven to increase the risk of secondary cancer. This can be traced by the mutagenic DNA adduct formation, for instance, N5-nitrogen mustard formamidopyrimidine (NM-Fapy-G). Therefore, it may serve as one of the secondary cancer biomarkers in patients receiving cyclophosphamide. Several NM-Fapy-G analysis methods employ Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) developed by experts. However, it was discovered not applicative for patients because cells and tissues were utilized as the biospecimens. Therefore, this summary is presented to emphasize the idea of adopting Dried Blood Spot (DBS) as the blood bio sampling method, DNA extraction and hydrolysis method that is suitable for enriching NM-Fapy-G adduct; and method that is proper for NM-Fapy-G analysis. Based on the literature study, DBS has been proven beneficial for this analysis; DNA can be extracted from the DBS cards using the QIAamp DNA Mini Kit. Therefore, research with a little bit of adjustment can be applied for NM-Fapy-G analysis.

CIRCULATING CELL-FREE DNA (CCFDNA) ISOLATION FROM PATIENTS WITH DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL): COMPARATIVE ANALYSIS OF COMMERCIAL KITS

Introduction Isolation of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) are useful methodologies for studies of neoplastic genetic material and can provide information about cancer heterogeneity, prognosis and minimal residual disease (MRD). Therefore, the recovery of ccfDNA in sufficient quantity and adequate purity is crucial to provide accurate and reproducible results. In this study, we investigate the efficiency of four different commercial kits to recover short fragments of DNA compatible with the ccfDNA. Methods Ten mL of total peripheral blood from seven patients with Diffuse Large B-Cell Lymphoma (DLBCL) were collected in EDTA and were immediately processed. Plasma was obtained by centrifugation at 1.900 x g for 10 min at 4 °C, followed by other centrifugation at 20.000 x g for 10 min at 4 °C to remove cellular debris. Aliquots of 1.0 mL of plasma were stored at -80 °C until ccfDNA extraction. The ccfDNA extraction was performed using the same samples by the following kits: QIAamp MinElute ccfDNA kit (CFDNA - Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (QBLOOD - Qiagen, Hilden, Germany), Illustra Blood GenomicPrep Mini Spin Kit (GE - GE Healthcare Bio-Sciences Corp, UK) and ccfDNA isolation kit Quick-ccfDNA Serum & Plasma Kit (ZYMO - Zymo Research, Irvine, USA) according to the manufacturer's recommendations. The ccfDNA obtained was eluted in 30 μL of elution buffer for the subsequent experiments. The measurement of the ccfDNA obtained for each kit was performed in a Qubit 2.0 fluorometer using Qubit dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, USA). The analysis of integrity, purity and the size of the fragments of DNA were performed in the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) equipment using the High Sensitivity DNA kit (Agilent Technologies, Germany). Results The results obtained for each commercial kit in the Qubit-analysis were: CFDNA (0.27 ng/uL), QBLOO D (0.26 ng/uL), ZYMO (0.03 ng/uL) and GE (0.02 ng/uL). The Bionalyzer eletrophoresis showed that CFDNA kit was able to recover two different fragments sizes of ccfDNA one of 360–380 bp (0.114 ng/uL) and other of 160-180 bp (1.808 ng/uL). The DNA recovered with QBLOOD revealed sizes of 360-380 bp (0.031 ng/uL) and 160-180 bp (0.079 ng/uL). Both kits, GE and ZYMO were not able to isolate fragments of DNA smaller than 10.380 pb. Conclusion We demonstrated that QIAamp MinElute ccfDNA kit (CFDNA) was capable to recovery high quantity of small fragments of DNA compatible with ccfDNA. This result supports the use of protocols based in magnetic beads in experiments working with ccfDNA. Additionally, we highlight the importance to verify the quality and not only the quantity of the DNA extracted to confirm the presence of ccfDNA.

Effectiveness of D,L-2-hydroxyisocaproic acid (HICA) and alpha-mangostin against endodontopathogenic microorganisms in a multispecies bacterial-fungal biofilm in an ex vivo tooth model.

To develop a defined multispecies root canal biofilm model ex vivo, and to perform viable compositional analysis following D,L-2-hydroxyisocaproic acid (HICA), alpha-mangostin, Calcicur® , and Odontopaste® exposure. Time-kill assays were conducted in vitro using HICA, alpha-mangostin, Calcicur® , Odontopaste® , and saline solution on the planktonic cultures of C.albicans, E.faecalis, L.rhamnosus, and S.gordonii. Human root dentine blocks were prepared (n=100) ex vivo, and multispecies suspensions containing each of 1.5×108 CFU/mL C.albicans, E.faecalis, L.rhamnosus, and S.gordonii in brain heart infusion (BHI) were incubated within the root canals for 21days. Canals (n=20/group) were then exposed to medicaments for 7days. Samples taken from the inner (first 0.1mm) and deeper (second 0.1mm) dentine by drilling with Ash Steel Burs No. 5 and No. 6, and residual roots were cultured in broth for 24h. Cell growth was detected by spectrophotometry and confirmed by culture on agar. The other set of inner dentine, deeper dentine, and residual root samples were sonicated, and then exposed with 50μM PMA before DNA was extracted using the QIAamp DNA mini kit. Real-time quantitative PCR was performed to determine the biofilm composition as well as the number of live and total cells remaining in the biofilm following each treatment. The OD data were analysed with Kruskal-Wallis and Friedman with Wilcoxon signed-rank test between and within groups, respectively, agar culture and qPCR data with Pearson chi-square with Mann-Whitney and Cochran with McNemar tests, respectively (p<.0001). Time-kill assays revealed that HICA and Calcicur® killed all planktonic organisms within 24h, whilst alpha-mangostin killed the organisms within 72h. However, Odontopaste® was a slow-killing agent: 10 cells of planktonic organisms survived after exposure to the agent for 7days. The ex vivo tooth model demonstrated that HICA and alpha-mangostin significantly inhibited the cell growth in all sampling depths (p<.0001). All species-specific data revealed the effectiveness of each medicament on the biofilm composition. D,L-2-hydroxyisocaproic acid and alpha-mangostin had antimicrobial activity against multispecies bacterial-fungal biofilms.

P–517 The relationship between mitochondrial gene CYB (MT-CYB) single nucleotide polymorphisms and male infertility

Abstract Study question Do single nucleotide polymorphisms of the mitochondrial gene CYB (MT-CYB) affect male fertility? Summary answer there was a significant association between male fertility and rs527236194, rs28357373, and rs41504845 single nucleotide polymorphisms of the mitochondrial gene CYB (MT-CYB). What is known already Male infertility can be occurred as a result of various factors. However, genetic factors are detected in 15% of male infertility cases and can be classified into two groups: chromosomal abnormalities and single gene mutations. Sperm mitochondrial DNA alterations may have serious effects on spermatogenesis, sperm motility and the ability of sperm to fertilize the oocyte. Mutations of the MT-CYB gene might lead to various disorders and deficiencies specially in complex III which might interrupt in the ATP production process. Study design, size, duration: 111 semen samples were included in this prospectively designed study which carried out between 2017 and August 2019. Participants/materials, setting, methods: This study carried out at the Department of Obstetrics and Gynecology at Saarland University, Germany. Samples were divided into 67 subfertile “cases” and 44 fertile “control” groups. After preparation of semen samples by density gradient centrifugation, nuclear and mitochondrial DNA (MT-DNA) was extracted using QIAamp DNA Mini Kit from QIAGEN. Thereafter, the MT-DNA was amplified using REPLI-g Mitochondrial DNA Kit from QIAGEN, followed by PCR and Sanger sequencing steps. Main results and the role of chance A total of 13 single nucleotide polymorphisms (SNPs) in the MT-CYB gene in each of the case and control groups were detected. Eight SNPs were non-synonymous variants including: rs2853508, rs28357685, rs41518645, rs2853507, rs28357376, rs35070048, rs2853506, and rs28660155 and five SNPs were synonymous variants: rs527236194, rs28357373, rs28357369, rs41504845, and rs2854124.Among these SNPs, three of them showed a significant difference in the genotype’s frequency test between sub-fertile and fertile groups: rs527236194 (T15784C; P = 0.0005), rs28357373 (T15629C; P = 0.0439), and rs41504845 (C15833T; P = 0.0038). For the allele’s frequency test, two SNPs were significant: rs527236194 (T15784C; P = 0.0014) and rs41504845 (C15833T; P = 0.0147). Limitations, reasons for caution The study population size Wider implications of the findings: A larger prospective study will be required to confirm the associations between these mitochondrial gene polymorphisms in MT-CYB (rs527236194, rs28357373, rs41504845) and male infertility and to clarify the definite effect of the mitochondrial genetic variations on male infertility. Trial registration number Not applicable

P–542 Relationship between the single nucleotides polymorphisms in Mitochondrial Nicotinamide Adenine Dinucleotide Hydride dehydrogenase (NADH) Subunit 4 gene (MT-ND4) 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 4 (MT-ND4)? Summary answer Our findings suggested that male infertility was correlated to rs2853495 and rs869096886 SNPs in MTND4. What is known already The rate of mutations in the mtDNA, the powerhouse of the cell, is high due to the lack of histones and DNA repair mechanisms. Therefore, mutations that occur in the mitochondrial genome, play a major role in some human genetic disorders. 15 - 30% of male infertility are related to genetic predisposition. Sperm containing defective mitochondria cannot effectively produce ATP and more likely to produce reactive oxygen species (ROS) and free radicals, thereby causing a defect in mtDNA, make trouble energy, and deteriorate motility and fertility. Study design, size, duration: A prospective study 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. Samples were divided into 68 subfertile and 44 fertile men. Mitochondrial DNA was extracted using a QIAamp DNA Mini Kit, after that the mtDNA was amplified by using REPLI-g Mitochondrial DNA Kit. Polymerase chain reaction (PCR) was used to amplify MT-ND4 gene. Then, samples were purified and sequenced using the Sanger method in the Microsynth Seq lab, Germany. Main results and the role of chance The genotypes frequencies of the study population showed a statistically significant association between rs2853495 G&amp;gt;A (Gly320Gly) and male infertility (P = 0.0351). Similarly, the allele frequency test showed that rs2853495 G&amp;gt;A (Gly320Gly) and rs869096886 A&amp;gt;G (Leu164Leu) were significantly associated with male infertility (adjusted OR = 2.616, 95% CI = 1.374 - 4.983, P = 0.0028; adjusted OR = 2.237, 95% CI = 1.245 - 4.017, P = 0.0073, respectively). On the other hand, no statistically significant difference was observed between the asthenozoospermia, oligozoospermia, teratozoospermia, asthenoteratozoospermia, oligoasthenoteratozoospermia, oligoteratozoospermia subgroups of subfertile males and the fertile ones. Limitations, reasons for caution The size number of the study samples. Wider implications of the findings: A larger prospective study will be required to confirm these associations of mitochondrial gene polymorphisms rs2853495 and rs869096886 in MT-ND4 and male infertility and to clarify the definite effect of the mitochondrial genetic variations on male infertility. Trial registration number Not applicable