Single Nucleotide Polymorphism Typing Research Articles

Review of SNP assays for disaster victim identification: Cost, time, and performance information for decision-makers.

In mass disaster events, forensic DNA laboratories may be called upon to quickly pivot their operations toward identifying bodies and reuniting remains with family members. Ideally, laboratories have considered this possibility in advance and have a plan in place. Compared with traditional short tandem repeat (STR) typing, single nucleotide polymorphisms (SNPs) may be better suited to these disaster victim identification (DVI) scenarios due to their small genomic target size, resulting in an improved success rate in degraded DNA samples. As the landscape of technology has shifted toward DNA sequencing, many forensic laboratories now have benchtop instruments available for massively parallel sequencing (MPS), facilitating this operational pivot from routine forensic STR casework to DVI SNP typing. Herein, we present the commercially available SNP sequencing assays amenable to DVI, we use data simulations to explore the potential for kinship prediction from SNP panels of varying sizes, and we give an example DVI scenario as context for presenting the matrix of considerations: kinship predictive potential, cost, and throughput of current SNP assay options. This information is intended to assist laboratories in choosing a SNP system for disaster preparedness.

Genetic Analyses of Rare ESBL ST628 Klebsiella pneumoniae Detected during a Protracted Nosocomial Outbreak in the United Kingdom.

Klebsiella pneumoniae (K. pneumoniae) cultures from a hospital-wide outbreak in the UK, which lasted for over 12 months, were sequenced. We sought to sequence and genetically characterise the outbreak strain. Antibiotic Susceptibility Testing (AST) was performed on 65 K. pneumoniae isolates saved from the outbreak. All isolates were sequenced using the Oxford Nanopore Technologies (ONT) MinION flowcell: 10 isolates, including the isolate with the earliest collection date in 2017, were additionally sequenced on the NovaSeq 6000 platform to build high-accuracy nanopore-illumina assemblies. Among the sequenced strains, 60 were typed as ST628. 96.6% (n = 58/60) ST628 strains harboured a large ~247-kb FIB(K) plasmid carrying up to 11 antimicrobial resistance genes, including the extended-spectrum beta-lactamase (ESBL) gene, blaCTX-M-15. Clonality between the outbreak isolates was confirmed using single nucleotide polymorphism (SNP) typing. The outbreak strains were phylogenetically related to clinical ST628 strains identified in 2012, 6 years prior to the outbreak. A rare ESBL K. pneumoniae K2 ST628 strain harbouring a multi-drug resistant (MDR) plasmid encoding the ESBL gene blaCTX-M-15 was detected across multiple independent wards during the protracted nosocomial outbreak. Surveillance of this strain is recommended to prevent future nosocomial outbreaks.

Detection of Single Nucleotide Polymorphisms of Circulating Tumor DNA by Strand Displacement Amplification Coupled with Liquid Chromatography.

The detection of multiple single nucleotide polymorphisms (SNPs) of circulating tumor DNA (ctDNA) is still a great challenge. In this study, we designed enzyme-assisted nucleic acid strand displacement amplification combined with high-performance liquid chromatography (HPLC) for the simultaneous detection of three ctDNA SNPs. First, the trace ctDNA could be hybridized to the specially designed template strand, which initiated the strand displacement nucleic acid amplification process under the synergistic action of DNA polymerase and restriction endonuclease. Then, the targets would be replaced with G-quadruplex fluorescent probes with different tail lengths. Finally, the HPLC-fluorescence assay enabled the separation and quantification of multiple signals. Notably, this method can simultaneously detect both the wild type (WT) and mutant type (MT) of multiple ctDNA SNPs. Within a linear range of 0.1 fM-0.1 nM, the detection limits of BRAF V600E-WT, EGFR T790M-WT, and KRAS 134A-WT and BRAF V600E-MT, EGFR T790M-MT, and KRAS 134A-MT were 29, 31, and 11 aM and 22, 29, and 33 aM, respectively. By using this method, the mutation rates of multiple ctDNA SNPs in blood samples from patients with lung or breast cancer can be obtained in a simple way, providing a convenient and highly sensitive analytical assay for the early screening and monitoring of lung cancer.

Risk prediction and function evaluation by T-cell epitope model and expression model of HLA-DPB1 mismatching in unrelated-donor hematopoietic stem cell transplantations

Objective: To evaluate the risk prediction and assessment function of HLA-DPB1 T-cell epitope (TCE) model and expression model in human leukocyte antigen (HLA)-matched unrelated hematopoietic stem cell transplantation (MUD-HSCT) with HLA-DPB1 mismatching. Methods: A total of 364 (182 pairs) potential MUD-HSCT donors and recipients confirmed by HLA high-resolution typing in Shaanxi Blood Center from 2016 to 2019 were analyzed retrospectively. Of the 182 recipients, there were 121 males and 61 females with an average age of (26.3±14.2) years. Of the 182 donors, there were 148 males and 34 females with an average age of (33.7±7.5) years. Polymerase chain reaction-sequence-based typing (PCR-SBT), next-generation sequencing (NGS) and polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSO) based on LABScan®3D platform were used for high-resolution typing of HLA-A, B, C, DRB1, DQB1, DPB1 gene, and PCR-SBT was used for single nucleotide polymorphism (SNP) typing. TCE model and expression model were used to predict and evaluate the HLA-DPB1 mismatch pattern and acute graft-versus-host-disease (aGVHD) risk. Results: A total of 26 HLA-DPB1 alleles and their 3'-UTR rs9277534 SNP genotypes were detected in this study population, and two new alleles HLA-DPB1*1052∶01 and HLA-DPB1*1119∶01 were found and officially named. The overall mismatch rate of HLA-DPB1 in MUD-HSCT donors and recipients was 90.66% (165/182). In TCE model, the HLA-DPB1 mismatch rates of permissible mismatch (PM) and non-permissible mismatch (non-PM) were 47.80% (87/182) and 42.86% (78/182), respectively. The non-PM in GvH direction was 13.73% (25/182), and which in HvG direction was 29.12% (53/182). A total of 73 pairs of donors and recipients in TCE model met the evaluation criteria of expression model. Among of TCE PM group, recipient DP5 mismatches accounted for 34.25% (25/73) were predicted as aGVHD high risk according to expression model. For the TCE non-PM group, both the recipient DP2 mismatches of 6.85% (5/73) and recipient DP5 mismatches of 10.86% (8/73) were predicted to be at high risk for aGVHD. Risk prediction by TCE model and expression model was 27.27% concordant and 16.97% unconcordant. Conclusions: TCE model and expression model are effective tools to predict aGVHD risk of MUD-HSCT. Comprehensive application of the two models is helpful to the hierarchical assessment of HSCT risk.

Whole Genome-Sequencing and Phylogenetic Analysis of Bacillus anthracis from 2019-2023 in Ningxia Hui Autonomous Region, China.

Since 2019, the incidence of anthrax in the Ningxia Hui Autonomous Region has increased significantly compared with previous years, so in this situation the anthrax in the Ningxia region not only had a detrimental impact on public health, but also inflicted significant economic repercussions. Therefore, we conducted a molecular epidemiological study of 20 strains from 2019-2023 isolates. This study investigated the origin of Bacillus anthracis and its genetic diversity. We conducted canonical single-nucleotide polymorphisms (CanSNPs) typing and whole genome sequencing based on the extracted nucleic acid of Bacillus anthracis. Based on the whole genome drafts, we studied the genomic characteristics of 20 isolates. Meanwhile, we performed phylogenetic studies based on genome-wide core single-nucleotide polymorphisms (SNPs) using MEGA's Maximum Likelihood (ML) method and core-genome-based multilocus sequence typing (cgMLST) of the core genomes of these strains using BioNumerics' minimum spanning tree (MST) model. The 20 isolates were categorized into sub-lineages A.Br.001/002, and comparative genomic analyses of these strains with other isolates from other parts of the world showed that the strains from Ningxia were correlated with isolates from Europe, Indonesia, Georgia (USA), and Beijing (China). For the 20 isolates in Ningxia, the genetic relationship of the isolates isolated from the same year or region was relatively close. The A.Br.001/002 subgroup was the dominant endemic strain in Ningxia. The genetic relationship and phylogenesis between isolates from Ningxia and strains from Europe and Indonesia suggest that anthrax spread around the globe through ancient trade routes.

Tracing the Evolutionary Pathways of Serogroup O78 Avian Pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) causes severe economic losses in the poultry industry, and O78 serogroup APEC strains are prevalent in chickens. In this study, we aimed to understand the evolutionary pathways and relationships between O78 APEC and other E. coli strains. To trace these evolutionary pathways, we classified 3101 E. coli strains into 306 subgenotypes according to the numbers and types of single nucleotide polymorphisms (RST0 to RST63-1) relative to the consensus sequence (RST0) of the RNA polymerase beta subunit gene and performed network analysis. The E. coli strains showed four apparently different evolutionary pathways (I-1, I-2, I-3, and II). The thirty-two Korean O78 APEC strains tested in this study were classified into RST4-4 (45.2%), RST3-1 (32.3%), RST21-1 (12.9%), RST4-5 (3.2%), RST5-1 (3.2%), and RST12-6 (3.2%), and all RSTs except RST21-1 (I-2) may have evolved through the same evolutionary pathway (I-1). A comparative genomic study revealed the highest relatedness between O78 strains of the same RST in terms of genome sequence coverage/identity and the spacer sequences of CRISPRs. The early-appearing RST3-1 and RST4-4 prevalence among O78 APEC strains may reflect the early settlement of O78 E. coli in chickens, after which these bacteria accumulated virulence and antibiotic resistance genes to become APEC strains. The zoonotic risk of the conventional O78 APEC strains is low at present, but the appearance of genetically distinct and multiple virulence gene-bearing RST21-1 O78 APEC strains may alert us to a need to evaluate their virulence in chickens as well as their zoonotic risk.

Development and validation of allele-specific PCR-based SNP typing in a gene on chromosome D03 conferring resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Upland cotton (Gossypium hirsutum) is the most important fiber crop for the global textile industry. Fusarium oxysporum f. sp. vasinfectum (FOV) is one of the most destructive soil-borne fungal pathogens in cotton. Among eight pathogenic races and other strains, FOV race 4 (FOV4) is the most virulent race in US cotton production. A single nucleotide polymorphism (SNP) in a glutamate receptor-like gene (GhGLR4.8) on chromosome D03 was previously identified and validated to confer resistance to FOV race 7, and targeted genome sequencing demonstrated that it was also associated with resistance to FOV4. The objective of this study was to develop an easy and convenient PCR-based marker assay. To target the resistance SNP, a forward primer for the SNP with a mismatch in the 3rd position was designed for both the resistance (R) and susceptibility (S) alleles, respectively, with addition of 20-mer T7 promoter primer to the 5' end of the forward primer for the R allele. The two forward primers, in combination with each of five common reverse primers, were targeted to amplify amplicons of 50-260bp in size with R and S alleles differing in 20bp. Results showed that each of three common reverse primers in combination with the two forward primers produced polymorphic markers between R and S plants that were consistent with the targeted genome sequencing results. The polymorphism was distinctly resolved using both polyacrylamide and agarose gel electrophoreses. In addition, a sequence comparative analysis between the resistance gene and homologous sequences in sequenced tetraploid and diploid A and D genome species showed that none of the species possessed the resistance gene allele, suggesting its recent origin from a natural point mutation. The allele-specific PCR-based SNP typing method based on a three-primer combination provides a fast and convenient marker-assisted selection method to search and select for FOV4-resistant Upland cotton.

Homology Identification and Cross-Contamination Analysis: A Method for Evaluating the Quality of Biological Samples Stored in a Biobank Using the Advanta Sample ID Genotyping Panel.

Biological samples are important resources for scientific research. These samples are stored in biobanks over years until needed, and some of them can never be retrieved if they are improperly stored, causing them to be wasted. Thus, they are priceless, and they should be used correctly and effectively. Sample quality substantially affects biomedical research results. However, sample misidentification or mix-up is common. It is necessary to establish quality standards for sample identification. In this study, we used the Advanta Sample ID genotyping panel to detect homology identification and cross-contamination. We compared the single-nucleotide polymorphism (SNP) typing results of two different samples and calculated the similarity score of homologous sample pairs and nonhomologous sample pairs. Through analysis, we obtained a similarity score cutoff point of 0.8620, which was an effective way to distinguish homology and nonhomology. Cross-contamination was detected in two sets of mixtures (STD8:STD6 and jj3:1-P) mixed at a series of special ratios. Sensitivity was dependent on the sample characteristics and mixing ratios. Finally, we assessed the effect of sample degradation degree on SNP genotyping and found that degraded samples with a minimal DNA integrity number of 1.9 had complete genotyping results. On the whole, this study shows that the Sample ID panel is reliable for homology identification and cross-contamination analysis. Moreover, this technology has promising further applications in biological sample quality control.

Factors associated with rituximab-mediated B cell depletion in ABO-incompatible adult living donor liver transplantation.

Pretransplant therapies such as rituximab and plasmapheresis have led to an increase in ABO-incompatible (ABOi) living donor liver transplantation (LDLT), thus helping to overcome organ shortages. This study evaluated the changes in anti-A/B titers and CD19 levels over time in patients undergoing ABOi LT and aimed to understand the effect of single-nucleotide polymorphisms (SNPs) in Fc gamma receptor (FcγR) on rituximab therapy. Two SNPs of FCGR2A (131H/R) and FCGR3A (158F/V) were identified. The clinical data on 44 patients who underwent ABOi LDLT between May 2019 and October 2021 at Seoul National University Hospital were reviewed retrospectively. Following desensitization with rituximab and subsequent LDLT, the anti-A/B titer recovered within 1 week, but decreased thereafter. The CD19 level increased at 3 months after LT. The genotyping data for FCGR3A (158F/V) indicated that two patients had the V/V genotype, and 42 had the F/V genotype. In the genotyping data for FCGR2A (131H/R), 21 patients had the H/H genotype, three had the R/R genotype, and 20 had the H/R genotype. However, there were no significant differences in anti-A/B and CD19 levels, bacteremia rates, T cell-mediated rejection, antibody-mediated rejection, or the survival rate among the FCGR2A types. There were significant changes in the anti-A/B titers and CD19 levels over time in each patient after ABOi LDLT. The difference in outcomes following LT according to the FcγR SNP type for rituximab was unclear. Further studies with larger sample sizes are needed to confirm the effect of FcγR SNPs on rituximab therapy.

Multiplex genotyping of SNPs in genomic DNA via hydrogel-based assay mediated with MutS and polyethylene glycol

The simultaneous genotyping of multiple single nucleotide polymorphisms (SNPs) in genomic DNA derived from organisms holds significant potential for applications such as precision medicine and food product authentication. However, conventional assay technologies including qPCR-based techniques, microarrays, and hydrogel-based assays face limitations in efficient multiplexing of SNPs, particularly for large-size DNA beyond kilobase scales, due to constraints in multiplex capability, specificity, or sensitivity. In this study, a hydrogel-based multiplex SNP genotyping platform specifically designed for genomic DNA is presented. This platform integrates the ligation detection reaction (LDR) and rolling circle amplification (RCA) techniques within a hydrogel-based multiplex sensing system, enabling adaptable and sensitive SNP genotyping for genomic DNA. To enhance the specificity of the assay, MutS protein and polyethylene glycol are introduced into the protocol, reducing the non-specific ligation and RCA reactions synergistically. With significant specificity improvement of over 10-fold, three types of SNPs within an artificially constructed ∼1000 bp double-stranded DNA (dsDNA) are successfully genotyped with double-digit picomolar sensitivity. Furthermore, the practical applicability of the developed process for the origin identification of raw materials is demonstrated by genotyping three types of SNPs within genomic DNA obtained from two closely related plant species, Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), containing ca. 3.5 gigabase genome size. Of notable significance, this study marks the premiere achievement in PCR-free multiplex genotyping of SNPs in genomic DNA using a single fluorophore.

The role of DNA in forensic science: A comprehensive review

Forensic genetics, leveraging molecular tools and scientific applications, has witnessed significant advancements in DNA analysis over the last three decades. These progressions have enhanced the discrimination power, speed, and sensitivity of DNA profiling methods, enabling the analysis of challenging samples. This article explores the significance of forensic genetics in criminal investigations, traces the historical evolution of DNA analysis techniques, and presents recent developments in the field. The article aims to provide a comprehensive understanding of the crucial role of forensic genetics in criminal investigations and sheds light on the latest trends and breakthroughs in this area. The evolution of DNA typing from ABO blood typing to the current standard of short tandem repeat (STR) analysis is discussed, along with alternative DNA analysis methods, such as Y-chromosome analysis and single nucleotide polymorphism (SNP) typing. Massively parallel sequencing (MPS) represents a groundbreaking advancement, enabling whole genome sequencing and addressing complex cases. The article also covers recent innovations, including DNA methylation analysis, body fluid identification, forensic DNA phenotyping, and genetic genealogy, highlighting their potential benefits in forensic investigations. Despite these advancements, standard STR profiling remains the gold standard due to its established protocols and databases. Ethical considerations regarding data privacy and cost implications are crucial as these technologies continue to progress in their pursuit of justice.

Correlation of ChREBP Gene Methylation with Pathological Characteristics of Type 2 Diabetes Mellitus.

The objective of this study is to investigate the expression of the carbohydrate response element binding protein (ChREBP) gene in type 2 diabetes mellitus (T2DM) and its correlation with pathological features.For obtaining and exploring the pathological features in patients, sixty T2DM patients (the research group) and thirty healthy controls (the control group) presented to our hospital between January 2019 and June 2019 were selected as the research participants. After DNA extraction from peripheral blood mononuclear cells (PBMCs) and modification of target gene methylation with bisulfite, differences in methylation were verified, and the correlation of ChREBP methylation level with T2DM pathological features and single nucleotide polymorphism (SNP) typing was discussed.According to the prediction results of UCSC Genome Browser Home, there were two CpG islands in the promoter region of the ChREBP gene, and the first exon was selected for research. The ChREBP methylation rate was statistically higher in the research group versus the control group (P < 0.05). Age, FPG, TC, and TG were confirmed by the multiple linear regression analysis to be correlated with the ChREBP methylation rate (P < 0.05). Finally, there was no difference in ChREBP methylation level between CT- and CC-type patients at rs17145750 and rs1051921 loci (P > 0.05).Peripheral blood ChREBP methylation is elevated in T2DM patients and is closely related to age, blood glucose, and blood-lipid level, which is expected to be a new direction for future T2DM diagnosis and treatment.

TRAIL Single Nucleotide Polymorphism in Type 2 Egyptian Diabetic Patients

TRAIL Single Nucleotide Polymorphism in Type 2 Egyptian Diabetic Patients

A novel strategy for screening mutations in the voltage-gated sodium channel gene of Aedes albopictus based on multiplex PCR-mass spectrometry minisequencing technology

BackgroundThe current prevention and control strategy for Aedes albopictus heavily relies on comprehensive management, such as environmental management and chemical control. However, the wide application of pyrethroids has facilitated the development of insecticide resistance, primarily via mutations in the voltage-gated sodium channel (VGSC) gene. This study aims to develop a novel strategy for detecting mutations in the VGSC gene in Ae. albopictus using multiplex PCR-mass spectrometry (MPCR-MS) minisequencing technology.MethodsWe established a new strategy for detecting mutations in the VGSC gene in Ae. albopictus using MPCR-MS minisequencing technology. MPCR amplification and mass probe extension (MPE) were first used, followed by single nucleotide polymorphism (SNP) typing mass spectrometry, which allows the simultaneous detection of multiple mutation sites of the VGSC gene in 96 samples of Ae. albopictus. A total of 70 wild-collected Ae. albopictus were used to evaluate the performance of the method by comparing it with other methods.ResultsThree target sites (1016, 1532, 1534) in the VGSC gene can be detected simultaneously by double PCR amplification combined with matrix-assisted laser desorption ionization–time-of-flight mass spectrometry, achieving a detection limit of 20 fg/μl. We applied this method to 70 wild-collected Ae. albopictus, and the obtained genotypes were consistent with the routine sequencing results, suggesting the accuracy of our method.ConclusionsMPCR-MS minisequencing technology provides a sensitive and high-throughput approach to Ae. albopictus VGSC gene mutation screening. Compared with conventional sequencing, this method is economical and time-saving. It is of great value for insecticide resistance surveillance in areas with a high risk of vector-borne disease.Graphical

Development of a POCT detection platform based on a locked nucleic acid-enhanced ARMS-RPA-GoldMag lateral flow assay

In this study, a novel genotyping point-of-care testing (POCT) rapid detection device, the locked nucleic acid (LNA)-amplification refractory mutation system (ARMS)-recombinase polymerase amplification (RPA)-GoldMag lateral flow assay (LFA) platform, was provided by mining and synthesis based on prior technology. Research methods based on system-integrated innovation and knowledge-integrated generation have become a new trend in technology development. Here, we exploit the combination of LNA-coupled ARMS-RPA and gold nanoparticle probe technology for detection signal amplification, thus pioneering a new tool for accurate, rapid, and cost-effective genotyping. We also performed SNP typing detection and clinical validation of this new assay platform using common glucose-6-phosphate dehydrogenase (G6PD) gene single nucleotide polymorphism (SNP) loci, and the results demonstrated the high sensitivity, specificity, stability, accuracy and feasibility of the LNA-ARMS-RPA-GoldMag lateral flow assay platform. It is hoped that this new technology will make a significant contribution to the field of POCT rapid diagnosis and aim to expand the application space, reflecting its clinical application value and development prospects.

Tumor Necrosis Factor-Alpha (+489 G/A) Polymorphism Can Predict the Response to Adalimumab in Chinese Han Patients With Ankylosing Spondylitis.

Studies investigating the association between single nucleotide polymorphisms (SNPs) of tumor necrosis factor-alpha (TNFα) and the efficacy of adalimumab (ADA) in ankylosing spondylitis (AS) therapy have reported conflicting results. We aimed to investigate the value of SNP typing of TNFα in predicting the efficacy of ADA in AS. Eighty patients with active AS who received ADA treatment were followed up for 24 weeks. Six known SNPs of TNFα (+489G/A, -238G/A, -308G/A, -857C/T, -863C/A, and -1031C/T) were subjected to the SNaPshot SNP typing method, which has been proven to be a reliable, efficient, and cost-effective method for detecting SNPs. The relationship between each SNP genotype and the therapeutic efficacy of ADA was analyzed. At the end of the 24-week follow-up, 58.8% of the patients with AS achieved Assessment of SpondyloArthritis International Society (ASAS) partial remission (PR), 67.5% of the patients achieved the criteria of an ASAS40response (40% improvement on indices), and 53.8% of the patients achieved Ankylosing Spondylitis Disease Activity Score (ASDAS) major improvement (MI). The univariate analysis showed that patients with AS carrying the TNFα +489 A allele were more likely to achieve ASAS-PR, ASAS40 response criteria, and ASDAS-MI after ADA treatment. In the multivariate regression analysis, the TNFα +489 A allele was an independent factor influencing the efficacy of ADA in treating AS (ASAS-PR odds ratio (OR) = 2.66, 95% confidence interval (CI) = 1.01-7.01; ASAS40 OR = 4.56, 95% CI = 1.39-15.00; ASDAS-MI OR = 3.31, 95% CI = 1.02-10.69). The patients carrying the TNFα +489 A allele may be more likely to experience better therapeutic efficacy and achieve the treatment target (ASAS-PR, ASAS40 response, or ASDAS-MI) after receiving ADA treatment. Detection of TNFα +489 G/A may predict the therapeutic efficacy of ADA, which can be used in clinical practice to tailor treatment for individual patients with AS. Further studies with larger sample sizes and longer follow-up periods with imaging evaluation are needed to verify our findings.

Implementation of routine genomic surveillance provided insights into a locally acquired outbreak caused by a rare clade of Salmonella enterica serovar Enteritidis in Queensland, Australia.

Salmonellosis is a significant public health problem globally. In Australia, Salmonella enterica serovar Enteritidis is one of the main causes of salmonellosis. This study reports how the implementation of routine genetic surveillance of isolates from human S. Enteritidis cases enabled identification of the likely source of an outbreak that occurred in a remote town in Far North Queensland, Australia. This study included patient, food and water samples collected during an outbreak investigation. S. Enteritidis of the novel sequence type 5438 was isolated from all seven patient samples and one bore water sample but not any of the food samples. Both whole-genome single nucleotide polymorphism (SNP) and core-genome multilocus sequence typing analysis revealed that S. Enteritidis isolated from outbreak-related patient samples and the bore water isolates clustered together with fewer than five SNP differences and ten allelic differences. This genetic relatedness informed the outbreak response team around public health interventions and no further cases were identified post-treatment of the bore water. This disease cluster was identified through the routine sequencing of S. Enteritidis performed by the state public health laboratory in an actionable time frame. Additionally, genomic surveillance captured a case with unknown epidemiological links to the affected community, ruled out a simultaneous outbreak in an adjacent state as the source and provided evidence for the likely source preventing further transmission. Therefore, this report provides compelling support for the implementation of whole-genome sequencing based genotyping methods in public health microbiology laboratories for better outbreak detection and management.

Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7-199 SNP and 3-109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44-70 with 28-38 cgMLST loci differences.Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

Phenotypic and genotypic discrimination of Francisella tularensis ssp. holarctica clades

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains.

In Silico Prediction and Molecular Docking of SNPs in NRP1 Gene Associated with SARS-COV-2.

Neuropilin-1 (NRP1) which is a main transmembrane cell surface receptor acts as a host cell mediator resulting in increasing the SARS-Cov-2 infectivity and also plays a role in neuronal development, angiogenesis and axonal outgrowth. The goal of this study is to estimate the impact of single nucleotide polymorphisms (SNPs) in the NRP1 gene on the function, structure and stabilization of protein as well as on the miRNA-mRNA binding regions using bioinformatical tools. It is also aimed to investigate the changes caused by SNPs in NRP1 on interactions with drug molecule and spike protein. The missense type of SNPs was analyzed using SIFT, PolyPhen-2, SNAP2, PROVEAN, Mutation Assessor, SNPs&GO, PhD-SNP, I-Mutant 3.0, MUpro, STRING, Project HOPE, ConSurf, and PolymiRTS. Docking analyses were conducted by AutoDock Vina program. As a result, a total of 733 missense SNPs were determined within the NRP1 gene and nine SNPs were specified as damaging to the protein. The modelling results showed that wild and mutant type amino acids had some different properties such as size, charge, and hydrophobicity. Additionally, their three-dimensional structures of protein were utilized for confirmation of these differences. After evaluating the results, nine polymorphisms rs141633354, rs142121081, rs145954532, rs200028992, rs200660300, rs369312020, rs370117610, rs370551432, rs370641686 were determined to be damaging on the structure and function of NRP1 protein and located in conserved regions. The results of molecular docking showed that the binding affinity values are nearly the same for wild-type and mutant structures support that the mutations carried out are not in the focus of the binding site, therefore the ligand does not affect the binding energy. It is expected that the results will be useful for future studies.