Genotyping Methods Research Articles

Distribution and Implications of Mycobacterium Tuberculosis Genotypes in Animals: A Comprehensive Review

This narrative review discusses the spread of Mycobacterium tuberculosis among animal populations, focusing on M. tuberculosis genotype distribution and frequency across different species. To develop comprehensive tuberculosis control strategies under the One Health approach, it is important to have an understanding of the genotypes of M. tuberculosis in animals. Animal infections are discussed, as well as the pathogenesis and epidemiology of M. tuberculosis. Various genotyping methods, including spoligotyping, MIRU-VNTR, and whole genome sequencing, are discussed in the review, emphasizing their role in understanding strain diversity and transmission dynamics. There are many factors influencing the distribution of M. tuberculosis genotypes in animals, including environmental conditions, host factors, human-animal interactions, and animal trade practices. It is clear from the review that the Euro-American lineage is widely distributed across animal species, with the Beijing genotype becoming increasingly prevalent in regions where it is prevalent in humans. The study highlights the need for more comprehensive genotyping studies as well as the development of better diagnostic tools. This will enhance our understanding of M. tuberculosis in animal populations. A conclusion to the review, emphasizes that standardizing genotyping protocols, utilizing whole genome sequencing, and fostering international collaboration are essential for improving our understanding of M. tuberculosis diversity in animals and developing targeted tuberculosis control strategies. The impact of Mycobacterium tuberculosis genotypes in animals is multi-faceted, affecting both public health and veterinary practices. Addressing these challenges requires a coordinated and comprehensive approach that considers the interconnectedness of human and animal health systems, fosters research, enhances surveillance, and implements effective control strategies.

Monitoring of pyrethroid resistance in Aedes aegypti: first report of double and triple kdr mutations in Buenos Aires Province

BackgroundDengue is an emerging disease in Argentina due to the colonization of Aedes aegypti, the mosquito vector. Buenos Aires Province is the biggest and most populated district in Argentina, suffering dengue outbreaks of growing magnitude. During epidemic periods, pyrethroid insecticides are used in this country to control adult mosquitoes. Pyrethroid resistance in dengue vectors has been reported worldwide, making it necessary to implement resistance management strategies. The voltage-gated sodium channel is the target site of pyrethroids. Mutations in the gene encoding this protein, called kdr mutations, are usually the molecular cause of pyrethroid resistance in insects. In Ae. aegypti from the Americas, three kdr substitutions were described: V410L, V1016I, and F1534C. The diagnostic of kdr mutations is recommended for the early detection of pyrethroid resistance as well as the consequent planning of evidence-based control policies.MethodsWe distributed ovitraps across 16 localities in Buenos Aires Province, collecting 22,123 eggs. A total of 522 mosquitoes were genotyped in positions 1016 and 1534 of voltage-gated channel using multiplex high-resolution melting and/or TaqMan probe methods. A subset of 449 samples was also genotyped by a singleplex high-resolution melting method developed ad hoc and/or Sanger sequencing.ResultsWe have documented, for the first time to our knowledge in the central region of Argentina, the presence of the 1016Ikdr + 1534Ckdr allele. Additionally, our study reports the first identification of the V410L mutation in central Argentina. These results underscore a growing trend of pyrethroid resistance in Ae. aegypti, fueled by the widespread use of these insecticides.ConclusionsWe detected 1016Ikdr + 1534Ckdr and 410Lkdr mutations in central Argentina for the first time and improved the processivity and accuracy of kdr genotyping methods. The results are both a tool for resistance monitoring and a sign of alarm to direct efforts towards finding sustainable methods for vector control to complement or replace pyrethroids. Joint efforts between academia and authorities to develop and implement public policies for vector control are a productive way to transfer scientific results for their application in public health.Graphical

Characterizing Escherichia coli carrying plasmid-mediated AmpC β-lactamases to optimize detection in a diagnostic laboratory setting.

Plasmid-mediated AmpC β-lactamases are a cause of acquired cephalosporin resistance in Gram-negative bacteria. However, consensus regarding the optimal detection method is yet to be achieved and varies depending on local epidemiology and laboratory capacity. We determined the acquired genotypic resistance mechanisms of 250 Escherichia coli isolates with a positive AmpC screen, defined as cefoxitin MIC ≥ 8 mg/L and a positive AmpC double- disc diffusion test, using in-house designed high-resolution melt PCR, detecting plasmid-acquired genes from the CIT and DHA families. A proportion of these isolates (n = 170, 68%) underwent further genotypic characterization using whole- genome sequencing (WGS). Of 250 isolates with a positive screening test, 72 (28.8%) were determined to carry an acquired AmpC gene. There was 100% concordance between PCR and WGS in the identification of acquired AmpC genes. The phenotypic criteria were then assessed to determine their utility in predicting acquired AmpC gene carriage. Criteria 1 (cefoxitin MIC > 8 mg/L plus ceftriaxone MIC > 1 mg/L and/or ceftazidime MIC > 1 mg/L) yielded a sensitivity of 93.1% and a specificity of 47.8%. Criteria 2 (cefoxitin MIC > 16 mg/L plus ceftriaxone MIC > 4 mg/L) had a sensitivity of 33.3% and a specificity of 98.9%. DHA genes, whose expression may be induced following antibiotic exposure, were present in 19% of isolates testing susceptible to ceftriaxone (MIC ≤ 1 mg/L) and were significantly more likely than CIT genes to be detected in susceptible isolates (P < 0.0001). These findings highlight the importance of using genotypic methods to detect acquired AmpC resistance in E. coli isolates that meet phenotypic screening criteria.IMPORTANCEDetection of transmissible AmpC resistance remains a challenging problem for diagnostic laboratories, especially in Escherichia coli where the expression of its intrinsic AmpC gene can result in phenotypic resistance patterns indistinguishable from plasmid-mediated resistance. In conjunction with whole- genome sequencing (WGS), we describe the development and performance of a novel melt-curve PCR to identify the two most prevalent plasmid-mediated AmpC gene families: CIT and DHA. We then describe phenotypic testing algorithms that incorporate this PCR and can differentiate these from non-acquired resistance in E. coli. It is important to distinguish these, not only to spare patients from unnecessarily being treated with infection control precautions, but also to identify plasmid-mediated genes, especially of the DHA family, that have been associated with inducible drug resistance to third- generation cephalosporins.

Genetic diversity insights from population genomics and machine learning tools for Nordic Arctic charr (Salvelinus alpinus) populations

Arctic charr (Salvelinus alpinus) is a salmonid species of high ecological and commercial value in the Holarctic region. Nevertheless, more information is needed about its underlying genetic diversity and population structure in the Nordics, especially regarding farmed populations. High-throughput sequencing was applied in three Arctic charr populations of anadromous or landlocked origin from Finland, Norway and Sweden. More specifically, the animals from the Swedish and Norwegian populations originated from a major egg supplier and producer, respectively. Furthermore, in the case of the Finnish population, the sampled animals originated from the only active conservation program for Arctic charr in the country with a potential interest in farming. Using double-digest restriction site-associated DNA sequencing (ddRAD-seq) on more than 500 fish, over 2000 single nucleotide polymorphisms (SNPs), both in the form of individual SNPs and as read haplotypes, were used to study the genetic diversity and structure of those populations. Genetic diversity metrics were similar between the Norwegian and the Swedish populations. However, substantially lower (40–50 %) genetic diversity was found in the Finnish population. Moreover, considerable genetic differentiation was implied between the studied populations as the mean fixation index (FST) was above 0.1 in all pairwise comparisons. All populations were easily discernible through either principal component analysis (PCA) or discriminant analysis of principal components (DAPC). In addition, unsupervised machine learning models such as K-means, Gaussian and Bayesian Gaussian mixtures were assessed for their ability to detect genetic clusters. A preceding dimensionality reduction step by PCA resulted in all three models, suggesting that the most probable number of clusters was three. Overall, our study affirmed the utility of the developed ddRAD-seq genotyping method and unveiled the genetic structure of the studied populations, both of which could contribute to their more efficient management by captive breeding.

Zootechnical characteristics of the emerging lines of stallions of the Yakut breed in the Oymyakonsky ulus of Yakutia

Purpose: zootechnical characteristics of the developing lines of Yakut breed stallions in the breeding farm "Agricultural production cooperative "Tonor" of the Oymyakon ulus of Yakutia.Materials and methods. The study of the exterior indicators of the stallions was carried out by taking body measurements according to the instructions of the All-Russian Research Institute of Horse Breeding. The use of genotypic assessment methods for animals along with classical zootechnical assessment methods for pedigree, productive traits (live weight and meat productivity of offspring, milk production, fertility of daughters) was supplemented by the use of the method of selecting offspring based on their adaptability to herd conditions.Results. Long-term selection work in the breeding farm of the Oymyakon ulus is completed by the formation of six lines of outstanding Yakut breed stallions. In terms of live weight, both stallions and mares of the Oymyakon horse surpass stallions and mares of the Yana, Kolyma and indigenous types. Oymyakon stallions (n=20) weighed on average 483,7±3,55 kg, while stallions of the indigenous types weighed 446,3±4,75 kg (n=34), Kolyma types 449,1±3,42 kg (n=87) and Yana types 469,4±3,27 kg (n=128). Oymyakon horse mares (n=165) also surpassed mares of the indigenous type (401,6±2,00 kg (n=341)), Kolyma type (432,2±1,13 kg (n=1063) and Yang type (431,0±1,16 kg (n=881)) in average live weight of 453,8±2,19 kg. According to measurements of head, neck, chest, croup, occiput and withers, chest and croup width and their indices, Oymyakon horse stallions and mares are practically no different from indigenous type horses. The founder of the line with the most numerous followers is the outstanding stallion Kyurzhet, born in 1946. He came from local Yakut horses of the former Bolshevik collective farm. His line continues on the Tonor farm through his two descendants: his grandson Chalay, born in 1981, live weight 515 kg, and his great-grandson Tenkelekh, born in 1992, live weight 490 kg. The farm produced Kyurzhet's great-great-great-grandson Chalay and 6 great-great-great-great-grandchildren, and now produces 11 continuers of the line (3 great-great-great-great-grandchildren and 8 great-great-great-great-great-grandchildren), a total of 18 continuers of his line have been and are being produced.

HIV tropism to chemokine coreceptors. Features of the definition, the current state

HIV infection is a significant cause of death worldwide, the number of people with HIV infection in the Russian Federation as of 2022 amounted to 1163818 people. The determination of HIV tropism is necessary for the appointment of drugs from the group of penetration inhibitors, and also opens up new opportunities in the prediction and analysis of HIV infection in a patient.Aim. To present a description and assess the current state of methods for determining HIV tropism, to summarize known information about the influence of HIV tropism on the course of the disease, to identify topical issues related to HIV tropism and requiring solutions.Materials and methods. A review of domestic and foreign sources devoted to methods for determining the prevalence and clinical significance of HIV tropism was carried out.Results. For the effective administration of CCR5 antagonist drugs, preliminary analysis is necessary to establish the tropism of HIV by genotypic or phenotypic methods. The use of CCR5 antagonists is not possible if HIV can use the CXCR4 coreceptor. CXCR4 – tropism of HIV is associated with the duration of the disease, a decrease in the number of CD4 cells, AIDS, and is a negative prognostic factor. Human mutations affecting coreceptors can affect the course of infection and susceptibility to HIV.Conclusion. The determination of HIV tropism is a useful analysis, the importance of which will increase in connection with the development of new drugs from the group of penetration inhibitors. To increase the availability of HIV tropism analysis in the Russian Federation, the creation of genotypic test systems is required. To create proprietary algorithms used in genotypic analysis, as well as laboratory testing and development of new effective drugs from the group of penetration inhibitors, it is necessary to develop a phenotypic test system. The small study of the influence of other regions of the env gene on HIV tropism, the study of HIV tropism to alternative coreceptors are urgent issues that need to be addressed.

GWAS for identification of genomic regions and candidate genes in vegetable crops.

Genome-wide association Studies (GWAS), initially developed for human genetics, have been highly effective in plant research, particularly for vegetable crops. GWAS is a robust tool for identifying genes associated with key traits such as yield, nutritional value, disease resistance, adaptability, and bioactive compound biosynthesis. Unlike traditional methods, GWAS does not require prior biological knowledge and can accurately pinpoint loci, minimizing false positives. The process involves developing a diverse panel, rigorous phenotyping and genotyping, and sophisticated statistical analysis using various models and software tools. By scanning the entire genome, GWAS identifies specific loci or single nucleotide polymorphisms (SNPs) linked to target traits. When a causal SNP variant is not directly genotyped, GWAS identifies SNPs in linkage disequilibrium (LD) with the causal variant, mapping the genetic interval. The method begins with careful panel selection, phenotyping, and genotyping, controlling for environmental effects and utilizing Best Linear Unbiased Prediction (BLUP). High-correlation, high-heritability traits are prioritized. Various genotyping methods address confounders like population structure and kinship. Bonferroni correction (BC) prevents false positives, and significant associations are shown in Manhattan plots. Candidate genes are identified through LD analysis and fine mapping, followed by functional validation. GWAS offers critical insights for enhancing vegetable crop breeding efficiency and precision, driving breakthroughs through advanced methods.

Antibiotic resistant Pseudomonas aeruginosa in dental unit waterline: a case study in Milan (Italy)

Abstract P.aeruginosa (Pa) is one of the most serious cause of healthcare-associated bacteremia, well-known for its antibiotic resistance (AR). Pa has often been found in dental unit water representing a great risk in public health. Aims are to evaluate the presence of Pa in dental unit (DU) water and its phenotypic and genotypic properties. Pa isolates derive from a monitoring of DU water in an Italian hospital (Milan). Samples were collected on Monday and on Friday, before the beginning and after the end of activities. Resistant PA strains were characterized by phenotypic method using E-test for 6 different classes of antibiotics, and by genotypic method with Whole-genome sequencing (WGS). Resistance genes were annotated with ResFinder v. 4.5 and Sequence Type (ST) was determined using the PA MLST database. 70/272 strains were identified as Pa and 21 of them were AR to one or more antibiotics. They were detected in 10/44 (22.7%) DUs and 2/4 (50%) control sinks (1-2000UFC/100mL). WGS was obtained for 18 strains. 17 belonged to serogroup O11 and ST667 was prevalent (8/18) followed by ST395 (4/18). At the ground floor ST395 was the most prevalent, followed by ST667 and 260, while, at the first floor, ST309 and 667 were in the two controls and ST667, 3245 and 260 in the other DUs. Resistant genes blaPAO, aph(3’)-IIb, catB7 conferring resistance to β-lactams, aminoglycosides and amphenicol, respectively, were observed in all isolates. Only ST3245 strain did not show resistance to Fosfomycin. Genotypic and phenotypic analyses present some discrepancies with the presence of AR genes and no antibiotic resistance detection (4/18) or resistance to other different classes of antibiotics (6/18). The MLST analyses showed a high diversity, with the presence of strains sharing different antibiotic resistance mechanisms in DU water. Detection and analyses by WGS can help to track dissemination of genetic resistance determinants and potential emergence of high-risk clones. Key messages • Phenotypic and genotypic analyses in DU water are needed to evaluate risk for human health, in addition to routine microbial monitoring. • WGS can help to track dissemination of genetic resistance determinants and potential emergence of high-risk clones.

Genotyping single nucleotide polymorphisms in homologous regions using multiplex kb level amplicon capture sequencing.

Single nucleotide polymorphisms (SNPs) in homologous regions play a critical role in the field of genetics. However, genotyping these SNPs is challenging due to the presence of repetitive sequences within genome, which demand specific method. We introduce a new, mid-throughput method that simplifies SNP genotyping in homologous DNA sequences by utilizing a combination of multiplex kb level PCR (PCR size 2.5k-3.5kb) for capturing targeted regions and multiplex nested PCR library construction for next-generation sequencing (Multi-kb level capture-seq). First of all, we randomly selected 7 SNPs in homologous regions and successfully captured 6-plex kb level amplicons (one of segments contains 2 SNPs, while the remaining segments each have only one SNP) in a single tube. And then, the amplification products were subjected to multiplex nested PCR for library construction and sequenced on Illumina platform. We tested this strategy using 600 amplicons from 100 samples and accurately genotyped 96.8% of target SNPs with a coverage depth of ≥ 15×. For the uniformity within the samples, over 66.7% (4/6) of the amplicons had a coverage depth above 0.2-fold of average sequencing depth. To validate the accuracy of this approach, we performed Ligase detection reaction PCR for genotyping the 100 samples, and found that the genotyping data was 97.71% consistent with our NGS results. In conclusion, we have developed a highly efficient and accurate method for SNP genotyping in homologous regions, which offers researchers a new strategy to explore the complex regions of genome.

Luteibacter sahnii sp. nov., A Novel Yellow-Colored Xanthomonadin Pigment Producing Probiotic Bacterium from Healthy Rice Seed Microbiome.

To explore the rice seed microbiome, our objective was to isolate novel strains of Xanthomonas, a plant-associated bacterium with diverse lifestyles. Four isolates, anticipated to be Xanthomonas based on morphological features of yellow colonies, were obtained from healthy rice seeds. Phylo-taxono-genomic analysis revealed that these isolates formed monophyletic lineages belonging to a novel species within the genus Luteibacter. Pairwise ortho Average Nucleotide Identity and digital DNA-DNA hybridization confirmed their distinct species status. We propose Luteibacter sahnii sp. nov. as a novel species, with PPL193T = MTCC 13290T = ICMP 24807T = CFBP 9144T as the type strain and PPL201, PPL552, and PPL554 as other constituent members. The fatty acid profile of the type strain is dominated by branched fatty acids like Iso-C15:0, consistent with other members of the genus. The novel species displays non-pathogenic attributes and exhibits plant probiotic properties, protecting rice plants from the leaf blight pathogen X. oryzae pv. oryzae. Production of Indole-3-Acetic Acid (IAA) and genomic regions encoding anti-microbial peptides emphasize its potential contributions to plant hosts. This study underscores the importance of employing a combination of phenotypic and genotypic methods in culturomics to enhance our understanding of rice seed microbiome diversity.

A novel machine-learning aided platform for rapid detection of urine ESBLs and carbapenemases: URECA-LAMP.

Pathogenic gram-negative bacteria frequently carry genes encoding extended-spectrum beta-lactamases (ESBL) and/or carbapenemases. Of great concern are carbapenem resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite the need for rapid AMR diagnostics globally, current molecular detection methods often require expensive equipment and trained personnel. Here, we present a novel machine-learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The platform consists of (i) an affordable device for sample lysis, LAMP amplification, and visual fluorometric detection; (ii) a LAMP screening panel to detect the most common ESBL and carbapenemase genes; and (iii) a smartphone application for automated interpretation of results. Validation studies on clinical isolates and urine samples demonstrated percent positive and negative agreements above 95% for all targets. Accuracy, precision, and recall values of the machine learning model deployed in the smartphone application were all above 92%. Providing a simplified workflow, minimal operation training, and results in less than an hour, this study demonstrated the platform's feasibility for near-patient testing in resource-limited settings.IMPORTANCEExtended-spectrum beta-lactamases (ESBL) and carbapenemases confer resistance to third-generation cephalosporins and carbapenems in pathogenic Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Conventional antimicrobial susceptibility testing is based on phenotypic methods, and results can take several days to be obtained. Current genotypic detection methods can be rapid but require expensive equipment and trained personnel. In this study, we present a novel machine learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The validation of the platform demonstrated percent positive and negative agreements above 95% for all targets. The newly developed platform provided a simplified workflow, minimal technical training, and results in less than an hour. This study demonstrated the platform's feasibility for rapid testing of ESBL and carbapenemases in bacteria and urine specimens.

Biologically-informed killer cell immunoglobulin-like receptor gene annotation tool.

Natural killer (NK) cells are essential components of the innate immune system, with their activity significantly regulated by Killer cell Immunoglobulin-like Receptors (KIRs). The diversity and structural complexity of KIR genes present significant challenges for accurate genotyping, essential for understanding NK cell functions and their implications in health and disease. Traditional genotyping methods struggle with the variable nature of KIR genes, leading to inaccuracies that can impede immunogenetic research. These challenges extend to high-quality phased assemblies, which have been recently popularized by the Human Pangenome Consortium. This article introduces BAKIR (Biologically informed Annotator for KIR locus), a tailored computational tool designed to overcome the challenges of KIR genotyping and annotation on high-quality, phased genome assemblies. BAKIR aims to enhance the accuracy of KIR gene annotations by structuring its annotation pipeline around identifying key functional mutations, thereby improving the identification and subsequent relevance of gene and allele calls. It uses a multi-stage mapping, alignment, and variant calling process to ensure high-precision gene and allele identification, while also maintaining high recall for sequences that are significantly mutated or truncated relative to the known allele database. BAKIR has been evaluated on a subset of the HPRC assemblies, where BAKIR was able to improve many of the associated annotations and call novel variants. BAKIR is freely available on GitHub, offering ease of access and use through multiple installation methods, including pip, conda, and singularity container, and is equipped with a user-friendly command-line interface, thereby promoting its adoption in the scientific community. BAKIR is available at github.com/algo-cancer/bakir.

An Inexpensive and Quick Method for Genotyping of HLA Variants Included in the Spanish Pharmacogenomic Portfolio of National Health System

The possibility of using the same genotyping technology (TaqMan) for all the genetic tests included in the new Spanish pharmacogenomics portfolio should enable the application of a multigenotyping platform to obtain a whole pharmacogenomics profile. However, HLA-typing is usually performed with other technologies and needs to be adapted to TaqMan assays. Our aim was to establish a set of TaqMan assays for correct typing of HLA-A*31:01, HLA-B*15:02, HLA-B*57:01, and HLA-B*58:01. Therefore, we searched for and selected SNVs described in different populations as surrogate markers for these HLA alleles, designed TaqMan assays, and tested in a set of samples with known HLA-A and HLA-B. HLA-A*31:01 was correctly typed with a combination of rs1061235 and rs17179220 (PPV 100%, 95% CI 84.6–100-%; NPV 100%, 95% CI 96.5–100.0%), HLA-B*15:02 with rs10484555 (PPV 100%, 95% CI 69.2–100.0%; NPV 100%, 95% CI 96.8–100.0%) and rs144012689 (PPV 100%, 95% CI 69.2–100.0%; NPV 100%, 95% CI 96.8–100.0%), and HLA-B*57:01 with rs2395029 (PPV 99.5%, 95% CI 72.9–99.3%; NPV 99.5%, 95% CI 98.3–100.0%). HLA-B*58:01 was typed using two allele-specific TaqMan probes mixed with a ß-Globin reference and treated as a genotyping assay (PPV 100.0%, 95% CI 81.5–100.0%; NPV 100%, 95% CI 96.8–100.0%). In conclusion, we demonstrated a clinically useful way to type HLA-A and HLA-B alleles included in the Spanish pharmacogenomics portfolio using TaqMan assays.

First Report of a Fatal Septicemia Case Caused by Vibrio metoecus: A Comprehensive Functional and Genomic Study

Abstract Background In recent years, we have witnessed an unprecedented increase in the incidence of vibriosis due to global warming. Vibrio metoecus is a recently described Vibrio cholerae-like species that has not been associated with septicemia death in humans. During follow-up of human vibriosis, we received a blood isolate from a patient with secondary septicemia who died a few hours after admission. Methods Phenotypic and genotypic methods failed to identify the isolate, which could only be identified by average nucleotide identity after genome sequencing. The isolate was subjected to in vitro and ex vivo assays, complemented by comparative genomics focused on the identification of unique genetic traits. Strains and genomes from the same and related species (V. cholerae and Vibrio mimicus) were used for analyses. Results The isolate was the only one able to resist and multiply in human serum. Its genome contained virulence genes shared with V. mimicus and/or V. cholerae, with those associated with sialic acid degradation within pathogenicity island 2 standing out. However, it also presented a unique gene cluster, flanked by a transposase gene, putatively related to surface polysaccharide pseudosialyzation. Conclusions We document the first case of death caused by septicemia due to V. metoecus and propose that the acquisition of surface pseudosialyzation genes explains the ability of certain isolates of this species to survive in blood. Our discovery underscores the urgent need to monitor and study newly emerging pathogenic species, as climate change may be facilitating their spread and increasing the risk of serious infections in humans.

ELISA genotyping of hepatitis B virus in China with antibodies specific for genotypes B and C

Hepatitis B virus (HBV) causes hepatitis B (HB) and distinct HBV genotypes can lead to different prognoses. However, HBV genotyping is rarely done in clinics, because the traditional method by PCR-based DNA sequencing is impractical for clinical diagnosis with tedious process and low success rate. Herein, we have established an ELISA-based genotyping method to quickly determine the HBV genotypes of HB patients in China. First, two commercial antibodies, 16D12 and 6H3 specific for HBV genotypes B and C respectively, are chosen as capture antibodies, since these two genotypes dominate in China. Then two home-made genotype-specific antibodies, B19 and C04, are used as the detection antibodies for genotypes B and C in sandwiched ELISA. The ELISA kit shows high sensitivity (> 95%) and specificity (> 95%) in detecting genotypes B and C of Chinese HB patients. Moreover, the ELISA kit has demonstrated higher success rate (98.7%) than PCR-based DNA sequencing (93.5%) and a commercial PCR-based genotyping kit (92.2%) for sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL. Such an advantage is more obvious for the sera with HBV DNA < 1000 IU/mL. The kappa analysis between the ELISA and PCR-based DNA sequencing results exhibits a kappa of 0.836, indicating a good correlation.

A Comprehensive Analysis of CSN1S2 I and II Transcripts Reveals Significant Genetic Diversity and Allele-Specific Exon Skipping in Ragusana and Amiatina Donkeys.

The αs2-casein is a phosphoprotein secreted in the milk of most mammals, and it is the most hydrophilic of all caseins. Contrary to genes found in ruminants, in donkeys two different encoding genes for donkey αs2-casein (CSN1S2 I and CSN1S2 II) have been identified. However, unlike in ruminants, the variability at these loci has not been characterized in detail in donkeys until now. In this study, we analyze the transcript profile of the donkey CSN1S2 I and CSN1S2 II genes, and we identify and describe the variability of these loci in the Ragusana and Amiatina breeds reared in Italy. The analysis of the CSN1S2 I Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) products and subsequent sequencing showed, in addition to correctly spliced mRNA, seven other minor mRNAs resulting from differential splicing events involving, in various combinations, entire exons (4, 5, 6, and 11), parts of exons (5' or 3' end of exon 17), or the recognition of intronic sequences as an exon (exon 12'). Similarly, the transcription analysis of the CSN1S2 II gene revealed a remarkable variability in splicing events, mainly concerning the alternative insertion of an extra exon 7 (named 7'); the first 33 bp of exon 13; or the alternative skipping of exons 9, 10, 11, 12, and 15, and their combinations. At the mRNA level for CSN1S2 I, seven SNPs were observed, five of which led to amino acid changes: p.T73>A, p.I109>V, p.I130>V, p.I146>T, and p.D217>Y. Similarly, nine SNPs were observed at the CSN1S2 II locus, seven of which are non-synonymous: p.L63>F, p.H70>Q, p.D90>N, p.129A>T, p.H131>Y, p.E144>G, and p.F157>S. In addition, the DNA sequencing of exon 17 and flanking introns of the CSN1S2 I gene revealed a G>A transition at the splice acceptor site of CSN1S2 I exon 17 (FM946022.1:c.375-1G>A), resulting in an allele-specific skipping of the first 15 nucleotides of this exon, which encode the peptide 176NKINQ180, and the recognition of an in-frame cryptic splicing acceptor site: arAACAAAATCAACCAG. A genotyping method based on restriction fragment length polymorphism (XbaI PCR-RFLP) was set up for this SNP. In the total population studied (105 Ragusana and 14 Amiatina donkeys), the A allele had a frequency of 0.2437 with no evidence of deviation from the Hardy-Weinberg equilibrium. This study adds new knowledge regarding the genetic variability of αs2-caseins in donkeys and may contribute significantly to the genetic improvement of milk production for this species.

Association of Polygenic Risk Score With Lifetime Risk of Developing Multiple Sclerosis in a Population-Based Birth-Year Cohort.

More than 200 genetic variants have been associated with multiple sclerosis (MS) susceptibility. However, it is unclear to what extent genetic factors influence lifetime risk of MS. Using a population-based birth-year cohort, we investigate the effect of genetics on lifetime risk of MS. In the Project Y study, we tracked down almost all persons with MS (pwMS) from birth year 1966 in the Netherlands. As control participants, we included non-MS participants from the Project Y cohort (born 1965-1967 in the Netherlands) and non-MS participants from the Amsterdam Dementia Cohort born between 1963 and 1969. Genetic variants associated with MS were determined in pwMS and control participants using genotyping or imputation methods. Polygenic risk scores (PRSs) based on variants and weights from the largest genetic study in MS were calculated for each participant and assigned into deciles based on the PRS distribution in the control participants. We examined the lifetime risk for each decile and the association between PRS and MS disease variables, including age at onset and time to secondary progression. MS-PRS was calculated for 285 pwMS (mean age 53.0 ± 0.9 years, 72.3% female) and 267 control participants (mean age 51.8 ± 3.2 years, 58.1% female). Based on the lifetime risk estimation, we observed that 1:2,739 of the women with the lowest 30% genetic risk developed MS, whereas 1:92 of the women with the top 10% highest risk developed MS. For men, only 1:7,900 developed MS in the lowest 30% genetic risk group, compared with 1:293 men with the top 10% genetic risk. The PRS was not significantly associated with age at onset and time to secondary progression in both sexes. Our results show that the lifetime risk of MS is strongly influenced by genetic factors. Our findings have the potential to support diagnostic certainty in individuals with suspected MS: a high PRS could strengthen a diagnosis, but especially a PRS from the lowest tail of the PRS distribution should be considered a red flag and could prevent misdiagnosing conditions that mimic MS.

Characterization of Chlorhexidine Resistance Among Clinical Isolates of Coagulase-Negative Staphylococcus Species in a Tertiary Care Center, Chennai.

Background Coagulase-negativeStaphylococci(CoNS) are potential pathogens and are often associated with healthcare-associated infections (HAIs). Chlorhexidine (CHX) is the most widely used antiseptic to reduce colonization and infection by allStaphylococci, including CoNS. Resistance to CHXamong CoNS has been observed over the past few years, consequent to its widespread use. Phenotypic tolerance or reduced susceptibility to CHX is conferred by plasmid-mediatedqacgroup of genes, mainlyqacA/Bandsmr, which cause activation of efflux pumps over the bacterial cell wall. This study aims to characterize the phenotypic and genotypic resistance exhibited by CoNS species against CHX. Methods After ethical approval, 148 consecutive, non-repetitive isolates of clinically significant CoNS species of hospitalized patients, isolated from blood samples and exudative specimens, were included in the study. Speciation was performed by conventional biochemical identification and automated methods. Antimicrobial susceptibility testing was performed by disc diffusion technique and for vancomycin by minimum inhibitory concentration (MIC) determination, as per Clinical Laboratory Standards Institute (CLSI) M-100 2023 guidelines. Methicillin resistance was detected using a cefoxitin disc. MIC for CHX was performed by agar dilution method; reduced susceptibility was considered when MIC to CHX ≥4 µg/mL. The simplex polymerase chain reaction (PCR) was carried out with suitable controls to detectqacA/Bandsmr. Statistical analysis was conducted to determine the association ofqacA/Bandsmrgenes with MIC of CHXin the study isolates. Results Fifteen different species of CoNS were obtained from clinical samples. A high percentage of resistance was observed against various classes of antibiotics. Methicillin resistance was observed in 69.6% (103/148) of isolates. Of 148 CoNS, 52.7% (78/148) of isolates exhibited reduced susceptibility to CHX with an MIC ≥4 µg/mL. These isolates exhibited a higher percentage of methicillin resistance (75.6%, 59/78). By PCR, 34.5% (51/148) of isolates carried either or both genes. GeneqacA/Bwas solely detected in 27.02% (40/148) of isolates, of which 14 were CHX-tolerant and the remaining 26 were CHX-susceptible. Genesmrwas solely detected in 4.1% (6/148) of isolates comprising three isolates each in CHX-tolerant and susceptible categories.There were 3.4% (5/148) of isolates that harbored bothgenes, of which only one isolate was CHX-susceptible, while the other four were CHX-tolerant. A proportion of isolates that were phenotypically tolerant to CHX did not carry either or both genes. A significant statistical association was found between reduced susceptibility to CHX and the presence of antiseptic resistance genesin the study isolates (p-value=0.033942). Conclusion To our knowledge, this is the first study from South India to investigate CHX resistance among CoNS using phenotypic and genotypic methods. The rise of antiseptic resistance among CoNS is an emerging threat to current infection control practices. The presence ofqacA/Bandsmrgenes, especially in CHX susceptible isolates, is concerning since these resistance genes are located on transferable plasmids, and the isolates can develop resistance eventually upon exposure to CHX.

Integrating targeted genetic markers to genotyping-by-sequencing for an ultimate genotyping tool.

New selection methods, using trait-specific markers (marker-assisted selection (MAS)) and/or genome-wide markers (genomic selection (GS)), are becoming increasingly widespread in breeding programs. This new era requires innovative and cost-efficient solutions for genotyping. Reduction in sequencing cost has enhanced the use of high-throughput low-cost genotyping methods such as genotyping-by-sequencing (GBS) for genome-wide single-nucleotide polymorphism (SNP) profiling in large breeding populations. However, the major weakness of GBS methodologies is their inability to genotype targeted markers. Conversely, targeted methods, such as amplicon sequencing (AmpSeq), often face cost constraints, hindering genome-wide genotyping across a large cohort. Although GBS and AmpSeq data can be generated from the same sample, an efficient method to achieve this is lacking. In this study, we present the Genome-wide & Targeted Amplicon (GTA) genotyping platform, an innovative way to integrate multiplex targeted amplicons into the GBS library preparation to provide an all-in-one cost-effective genotyping solution to breeders and research communities. Custom primers were designed to target 23 and 36 high-value markers associated with key agronomical traits in soybean and barley, respectively. The resulting multiplex amplicons were compatible with the GBS library preparation enabling both GBS and targeted genotyping data to be produced efficiently and cost-effectively. To facilitate data analysis, we have introduced Fast-GBS.v3, a user-friendly bioinformatic pipeline that generates comprehensive outputs from data obtained following sequencing of GTA libraries. This high-throughput low-cost approach will greatly facilitate the application of DNA markers as it provides required markers for both MAS and GS in a single assay.

A rapid economical multiplex PCR-RFLP method for molecular detection and genotyping of Giardia duodenalis clinical isolates

Giardia duodenalis is a common cause of diarrheal illness in regions with limited resources. The demand for rapid and cost-effective detection and genotyping methods in large-scale epidemiological studies and clinical diagnostics is imperative. Hence, we developed a multiplex PCR-RFLP technique targeting the tpi gene of G. duodenalis. The assay successfully screened G. duodenalis positive clinical samples (6.33 %; 36/565). It was also able to categorize the isolates into assemblages A (41.66 %; 13/36) and B (58.33 %; 23/36), as well as into subassemblages: AI (13.8 %; 5/36), AII (27.77 %; 10/36), BIII (36.11 %; 15/36) and BIV (22.22 %; 8/36). High diagnostic sensitivity (94.2 %), specificity (100 %) and accuracy (97.1 %) of the PCR assay were obtained, indicating its reliability for diagnosing giardiasis. Notably, the assay demonstrated close concordance with microscopy (κ=0.85) and reference PCR (κ=0.98) results. The optimized method offers a cost-effective and rapid approach for G. duodenalis detection and genotyping, convenient for epidemiological studies and clinical diagnostics.