The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Analysis of Tandem Repeats in Short-Read Sequencing Data: From Genotyping Known Pathogenic Repeats to Discovering Novel Expansions.

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genotyping known pathogenic tandem repeat loci Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy Support Protocol 1: Installation of tools and ExpansionHunter catalog modification Basic Protocol 2: Performing genome-wide genotyping of tandem repeats Basic Protocol 3: Discovering de novo tandem repeat expansions Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles.

ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria.

Background: Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria. Methods: Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology. Result: In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32). Conclusion and recommendations: Here a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

Genetic marker: a genome mapping tool to decode genetic diversity of livestock animals.

Genotyping is the process of determining the genetic makeup of an organism by examining its DNA sequences using various genetic markers. It has been widely used in various fields, such as agriculture, biomedical and conservation research, to study genetic diversity, inheritance, the genetic basis of disease-associated traits, evolution, adaptation, etc., Genotyping markers have evolved immensely and are broadly classified as random markers (RFLP, RAPD, AFLP, etc.) and functional markers (SCoT, CDDP, SRAP, etc.). However, functional markers are very limited in genotype studies, especially in animal science, despite their advantages in overcoming the limitations of random markers, which are directly linked with phenotypic traits, high specificity, and similar logistic requirements. The current review surveyed the available random and functional markers for genotyping applications, focusing on livestock including plant and microbe domains. This review article summarises the application, advantages, and limitations of developed markers and methods for genotyping applications. This review aims to make the reader aware of all available markers, their design principles, and methods, and we discuss the marker inheritance patterns of RLFP and AFLP. The review further outlines the marker selection for particular applications and endorses the application of functional markers in genotyping research.

Phylogenetic evaluation and genotypic identification of burn-related Pseudomonas aeruginosa strains isolated from post-burn human infections during hospitalization.

Cutaneous burn trauma, compromise of dermal layers and immune defense system is a physical and fiscal burden on healthcare systems. Burn-wound infections are a serious complication of thermal-injury and contribute significantly to care burden. After burn-induced trauma, sepsis by Pseudomonas aeruginosa impairs patient recovery and contributes to mortality and morbidity. Past studies show positive correlation between detection of Pseudomonas species and healing-impaired traumatic wounds. P. aeruginosa is a resilient opportunistic human pathogen and a nosocomial agent involved in pathology of healing-impaired wounds, especially in burn-patients. Expansive array of virulence determinants have resulted in gentamicin- and silver-resistant P. aeruginosa outbreaks. Knowledge of molecular dynamics and phylogeny of P. aeruginosa associated with burn-wounds is limited. Therefore, we conducted whole-genome sequencing for genotyping and phylogenetic analysis of P. aeruginosa burn-associated strains (n = 19) isolated from 7 burn cases during hospitalization. Comparison of genetic features in P. aeruginosa strains in the core genome and mobilome detected genetic variations within some clonal infections overtime. Genetic variations were observed among different burn cases, with some features identified in severe lung infections. Polyclonal infections were also observed, with differing genotypes and virulence potentials, highlighting the importance of reasoned sampling of isolates for clinical testing.

Retrospective analysis of HPV infection: Cotesting and HPV genotyping in cervical cancer screening within a large academic health care system.

BACKGROUND: In 2019, the American Society for Colposcopy and Cervical Pathology introduced fundamental shifts toward "risk-based" guidelines, with human papillomavirus (HPV) genotyping as a principal test for investigating squamous intraepithelial lesions. This study aims to provide practice-based evidence and supplement the updated guidelines by investigating HPV demographic distribution and uncovering the pathological features of high-grade squamous intraepithelial lesions (HSILs) caused by high-risk HPV (hrHPV) subtypes. METHODS: Patients who underwent Papanicolaou screening and HPV testing in two hospital systems over the course of 4 years were recruited. The cytology results were categorized on the basis of the 2014 Bethesda classification. DNA sequences of 14 types of hrHPV were detected by Aptima test. The histological features of HSILs caused by different subtypes were compared between biopsies and excisions. RESULTS: A total of 63,709 cases were included. The HPV prevalence was 14.70%, predominantly in the 30 to 39-year-old age group, with slightly higher rates observed in African Americans. There was no significant racial distribution difference between HPV 16/18/45 and other types. HPV 16/18/45 infection was directly correlated with the severity of abnormal cytology, although the other subtypes were the major causes of cytological abnormalities. The trend for HPV prevalence was consistent across calendar years, and was associated with 8.77% negative for intraepithelial lesion or malignancy, 30.46% atypical squamous cell of undetermined significance, 64.62% low-grade squamous intraepithelial lesion, 66.75% atypical squamous cell-cannot exclude a high-grade squamous intraepithelial lesion, and 91.80% HSIL. Furthermore, 29.09% of HSILs associated with other subtypes were not detectable on subsequent resections. CONCLUSIONS: Given the HPV demographic distribution and the histological features of HSILs caused by different subtypes, cotesting with reflex HPV genotyping in specific populations, or expanding the subtypes in the primary HPV screening test, should be considered.

Corrigendum: Genomic profiling of methicillin-sensitive Staphylococcus aureus (MSSA) isolates in Kuwait hospitals.

[This corrects the article DOI: 10.3389/fmicb.2024.1361217.].

Megaselia scalaris and Senotainia tricuspis Infesting Apis mellifera: Detection by Quantitative PCR, Genotyping, and Involvement in the Transmission of Microbial Pathogens.

The Megaselia scalaris and Senotainia tricuspis parasitoid flies of the honeybee Apis mellifera were found to infest apiaries of different European and Mediterranean countries but their prevalence and impact on apiary health are little known. Therefore, in this study, quantitative PCR (qPCR)-based methods were developed for their rapid detection directly in hive matrices. The newly developed qPCR assays were targeted at the mitochondrial cytochrome oxidase subunit I (COI) gene for the M. scalaris and the cytochrome B (cytB) gene for the S. tricuspis. The tests were preliminarily applied to 64 samples of adult honeybees and hive debris collected in the Abruzzo and Molise regions, Central Italy, and the Republic of Kosovo showing that both flies occur in the two countries and more frequently in Italy. The positive apiaries in Italy were re-sampled by capturing viable forager bees and isolating emerging flies to carry out the genotyping and analyses aimed at defining if these flies can transmit honeybee pathogens. Genotyping based on the COI and cytB gene sequencing for M. scalaris and S. tricuspis, respectively, identified one S. tricuspis genotype and diverse genotypes of M. scalaris highly similar to those from distant countries. Some fly isolates harbored the DNA or RNA of honeybee microbial pathogens Paenibacillus larvae, deformed wing viruses A and B (DWVA and B), black queen cell virus (BQCV), chronic paralysis virus (CBPV), and Nosema ceranae. The results indicated that these parasites should be efficiently controlled in apiaries by using rapid detection methods to facilitate the large screening studies and early detection.

Field-Applicable Loop-Mediated Isothermal Amplification for the Detection of Seven Common Human Papillomavirus Subtypes.

Persistent HPV infection is a major risk factor for the subsequent development of cervical cancer. LAMP is simple and suitable for field detection in the resource-limited settings. In this study, hydroxy naphthol blue (HNB)-based visual LAMP and evagreen-based fluorescent LAMP coupled with a microfluidic chip (LAMP-chip) were established for the field detection of seven subtypes of HPV. The analytical sensitivity was 19-233 copies/reaction. The overall clinical sensitivity was 97.35% for visual LAMP and 98.23% for LAMP-chip. Both LAMP assays exhibited 100% specificity and were completed in less than 50 min. Additionally, both assays did not require complicated nucleic acid extraction and purification steps. A complete quality control monitoring system (including internal control, positive quality control and negative control) in the LAMP assays further ensured the credibility of the results. Our findings demonstrated that the proposed LAMP assays have the potential to be applied in the testing of common HPV DNA in field investigations (visual LAMP) or within communities and primary health centers (LAMP-chip).

Sympatric diversity pattern driven by the secondary contact of two deeply divergent lineages of the soybean pod borer Leguminivora glycinivorella.

The soybean pod borer, Leguminivora glycinivorella (Matsumura), is an important tortricid pest species widely distributed in most parts of China and its adjacent regions. Here, we analyzed the genetic diversity and population differentiation of L. glycinivorella using diverse genetic information including the standard cox1 barcode sequences, mitochondrial genomes (mitogenomes), and single-nucleotide polymorphisms (SNPs) from genotyping-by-sequencing. Based on a comprehensive sampling (including adults or larvae of L. glycinivorella newly collected at 22 of the total 30 localities examined) that covers most of the known distribution range of this pest, analyses of 543 cox1 barcode sequences and 60 mitogenomes revealed that the traditionally recognized and widely distributed L. glycinivorella contains two sympatric and widely distributed genetic lineages (A and B) that were estimated to have diverged ∼1.14 million years ago during the middle Pleistocene. Moreover, low but statistically significant correlations were recognized between genetic differentiation and geographic or environmental distances, indicating the existence of local adaptation to some extent. Based on SNPs, phylogenetic inference, principal component analysis, fixation index, and admixture analysis all confirm the two divergent sympatric lineages. Compared with the stable demographic history of Lineage B, the expansion of Lineage A had possibly made the secondary contact of the two lineages probable, and this process may be driven by the climate fluctuation during the late Pleistocene as revealed by ecological niche modeling.

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-3.5 kb) 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.

Enhancing prediction accuracy of foliar essential oil content, growth, and stem quality in Eucalyptus globulus using multi-trait deep learning models.

Eucalyptus globulus Labill., is a recognized multipurpose tree, which stands out not only for the valuable qualities of its wood but also for the medicinal applications of the essential oil extracted from its leaves. In this study, we implemented an integrated strategy comprising genomic and phenomic approaches to predict foliar essential oil content, stem quality, and growth-related traits within a 9-year-old breeding population of E. globulus. The strategy involved evaluating Uni/Multi-trait deep learning (DL) models by incorporating genomic data related to single nucleotide polymorphisms (SNPs) and haplotypes, as well as the phenomic data from leaf near-infrared (NIR) spectroscopy. Our results showed that essential oil content (oil yield) ranged from 0.01 to 1.69% v/fw and had no significant correlation with any growth-related traits. This suggests that selection solely based on growth-related traits did n The emphases (colored text) from revisions were removed throughout the article. Confirm that this change is fine. ot influence the essential oil content. Genomic heritability estimates ranged from 0.25 (diameter at breast height (DBH) and oil yield) to 0.71 (DBH and stem straightness (ST)), while pedigree-based heritability exhibited a broader range, from 0.05 to 0.88. Notably, oil yield was found to be moderate to highly heritable, with genomic values ranging from 0.25 to 0.60, alongside a pedigree-based estimate of 0.48. The DL prediction models consistently achieved higher prediction accuracy (PA) values with a Multi-trait approach for most traits analyzed, including oil yield (0.699), tree height (0.772), DBH (0.745), slenderness coefficient (0.616), stem volume (0.757), and ST (0.764). The Uni-trait approach achieved superior PA values solely for branching quality (0.861). NIR spectral absorbance was the best omics data for CNN or MLP models with a Multi-trait approach. These results highlight considerable genetic variation within the Eucalyptus progeny trial, particularly regarding oil production. Our results contribute significantly to understanding omics-assisted deep learning models as a breeding strategy to improve growth-related traits and optimize essential oil production in this species.

Practice Patterns and Challenges in Managing Inherited Retinal Diseases Across Asia-Pacific: A Survey from the APIED Network.

PURPOSE: The objective of this paper is to shed light on the current landscape of genotyping practices, phenotyping practices and availability of essential vision rehabilitation management for inherited retinal diseases (IRD) in the Asia-Pacific (APAC) Region. METHODS: The 62-item questionnaire was distributed electronically via email. The questions covered five domains: (1) structure of the IRD service and registry/database; (2) genotyping practices; (3) genetic counselling; (4) deep phenotyping practices; (5) low-vision rehabilitation services. RESULTS: The survey was completed by 36 of 45 centres in twelve countries and regions in APAC. Among these centres, 42 % reported managing more than 1000 patients. Notably, 39 % of centres lack an IRD database or registry, and 44 % of centres have tested less than one-quarter of their IRD patients. The majority of centres (67 %) do not have genetic counsellors. While there was consistency in the imaging-based investigations, there was marked heterogeneity for functional testing using electrophysiology and formal perimetry. Only 34 % of centres confirmed the availability of access to low-vision assistive devices. CONCLUSIONS: This study reveals several critical gaps in managing IRDs in the APAC region. These include the lack of IRD database/registry in one-third of centres, a substantial proportion of patients remaining genetically undiagnosed, and limited availability of genetic counsellors. The findings also underscore a need to harmonise investigations for evaluating retinal function and identify areas for improvement in the provision of low-vision rehabilitation services.

Prevalence and genetic diversity of Giardia duodenalis in sheep from large-scale farms in Southern Xinjiang, China.

Giardia duodenalis is a significant zoonotic parasite. In this study, 767 fresh fecal samples were collected randomly from six large-scale sheep farms in Southern Xinjiang, China. Initially, G. duodenalis was screened using PCR at the SSU rRNA gene. Positive samples then underwent PCR amplification at the bg, gdh, and tpi genes. The prevalence of G. duodenalis in sheep was 17.5% (134/767), with the highest prevalence observed in the 3-6 months age group at 26.8% (56/209) and the lowest in the over 12 months age group at 6.8% (14/205). Among the 134 positive samples, only Assemblage E was identified. A total of 106, 92, and 98 sequences of G. duodenalis were obtained at the gdh, tpi, and bg genes, respectively. Fourteen isolates of G. duodenalis were successfully amplified at all three genes, resulting in nine G. duodenalis multilocus genotypes (MLG) named MLG E1-MLG E14, indicating high genetic diversity. In conclusion, G. duodenalis infection in sheep from large-scale farms is common in Southern Xinjiang, China, showing geographical regional distributions and genetic diversity.

Distribution of 14 High-Risk HPV Types and p16/Ki67 Dual-Stain Status in Post-Colposcopy Histology Results: Negative, Low- and High-Grade Cervical Squamous Intraepithelial Lesions.

BACKGROUND: Determining the distribution of high-risk human papillomavirus (HR-HPV) types in histologic low-(LSIL) and high-grade (HSIL/CIN2+) squamous intraepithelial lesions through a diagnostic process in a cervical cancer prevention provides one of the key etiological factors behind further progression and persistence. Incorporating novel high-grade cervical lesion biomarkers such as p16/Ki67 dual staining (DS) alongside HPV typing has become important in detecting cervical precancers. METHODS: Among 28,525 screening tests and 602 histology results, 559 cases with HR-HPV and histology results obtained from colposcopic biopsy were retrospectively analyzed, together with DS status. The χ2 test with Bonferroni correction evaluated the differences in HR-HPV type prevalence and DS positivity across three histologic study groups. RESULTS: A statistically significant difference in the prevalence of HPV 16 was observed between negative and HSIL/CIN2+ (p = 0.00027) groups, as well as between the LSIL/CIN1 and HSIL/CIN2+ groups (p = 0.00041). However, no significant difference was found between the negative and LSIL/CIN1 groups. Similarly, the DS positivity difference was significant between the negative and HSIL/CIN2+ (p < 0.0001) and between the LSIL/CIN1 and HSIL/CIN2+ groups (p < 0.0001), but there was no significant difference between the negative and LSIL/CIN1 groups. CONCLUSIONS: The study highlights the heterogeneous nature of HPV-related cervical pathologies, and the distinct risks associated with different cervical lesion grades, emphasizing the importance of HR-HPV type distribution and DS status.

Salivary glands - a new site of Helicobacter pylori occurrence.

OBJECTIVE: The role of Helicobacter pylori (Hp) in the pathological processes of the gastric mucosa is well understood. Decreasing trend in successful eradication of HP from the stomach was observed in last years. This lack of succes is mainly caused by increasing ATB resistance. Nevertheless other possible causes of this phenomenon are being explored. Thus, many studies have focused on the search for extragastric reservoirs as potential sources of persistence or reinfection after successful Hp eradication. The pathological potential of Hp at these localities has also been studied. METHODS: Our study aimed to determine the presence of Hp inside the salivary glands ductal system through its detection from sialolites. Subsequently, we tried to prove the possible ability of Hp to penetrate the salivary gland parenchyma by detecting Hp from the tissue of salivary tumors. Concrements and salivary tumor tissue samples were collected using sialendoscopy or standard surgery, and Hp detection and genotyping were performed through PCR. RESULTS: Hp was detected in 68.3% of the sialopathy samples. VacA S1AM1 was the most common genotype. CagA-positive genotype represented only 34% of the total number of positive samples. CONCLUSION: Our findings of Hp positivity in concrements provide compelling evidence of Hp presence in the ductal system of salivary glands. Confirmation of Hp presence in tumor tissue suggests its potential ability to infiltrate the gland's parenchyma. Further research is needed to confirm Hp's ability to cause local infection, as well as the possible causal association between Hp presence in the studied region, sialolithiasis, and salivary gland tumors.

Genotyping Error Detection and Customised Filtration for SNP Datasets.

A major challenge in analysing single-nucleotide polymorphism (SNP) genotype datasets is detecting and filtering errors that bias analyses and misinterpret ecological and evolutionary processes. Here, we present a comprehensive method to estimate and minimise genotyping error rates (deviations from the 'true' genotype) in any SNP datasets using triplicates (three repeats of the same sample) in a four-step filtration pipeline. The approach involves: (1) SNP filtering by missing data; (2) SNP filtering by error rates; (3) sample filtering by missing data and (4) detection of recaptured individuals by using estimated SNP error rates. The modular pipeline is provided in an R script that allows customised adjustments. We demonstrate the applicability of the method using non-invasive sampling from the Asiatic wild ass (Equus hemionus) population in Israel. We genotyped 756 samples using 625 SNPs, of which 255 were triplicates of 85 samples. The average SNP error rate, calculated based on the number of mismatching genotypes across triplicates before filtration, was 0.0034 and was reduced to 0.00174 following filtration. Evaluating genetic distance (GD) and relatedness (r) between triplicates before and after filtration (expected to be at the minimum and maximum respectively) showed a significant reduction in the average GD, from 58.1 to 25.3 (p = 0.0002) and a significant increase in relatedness, from r = 0.98 to r = 0.991 (p = 0.00587). We demonstrate how error rate estimation enhances recapture detection and improves genotype quality.

Novel genotyping definition and molecular characteristics of canine circovirus in China.

Canine circovirus (CCV) has been detected globally, but its genotyping remains unevenly characterized. To comprehend the evolution and genotyping of CCV, 887 rectal swabs of dogs were collected during 2018-2023. According to p-distance/frequency histograms and phylogenetic trees based on complete genome sequences, the 21 newly obtained Chinese and 84 reference CCV strains were mainly divided into 7 subtypes (CCV-1a, CCV-1b, CCV-1c, CCV-1d, CCV-1e, CCV-2a, and CCV-2b). Among the 21 newly obtained CCVs, 9 strains belonged to CCV-1d, 2 strains belonged to CCV-1b, and the remaining strains belonged to CCV-1c. Recombination analysis indicated that recombination events occurred between CCV strains of different subtypes, hosts, and countries. The CCV capsid protein features 19 variable sites, with 2 sites (T58Q, P239A) displaying regional specificity and 3 (Q57T, E150Q, and T195Q) manifesting subtype specificity. Therefore, this genotyping analysis provides a reference for the molecular characteristics of CCV strains found globally.

Isolation of genetically distinct strains within the same species during treatment of MAC pulmonary disease.

BACKGROUND: Research on isolating genetically different strains within the same species in patients undergoing treatment for Mycobacterium avium complex (MAC) pulmonary disease (PD) is limited. We investigated the frequency of genetically distinct strains identified within the same species among on-treatment isolates compared with pre-treatment isolates throughout the course of MAC-PD treatment. RESEARCH QUESTION: What is the frequency of genetically distinct strains identified within the same species among pre- and on-treatment isolates in patients with MAC-PD? STUDY DESIGN AND METHODS: We serially collected pre- and on-treatment clinical isolates from patients with MAC-PD treated for over one month from November 2019 to October 2022 at a tertiary hospital in South Korea. We utilized multilocus sequence typing (MLST) genotypic analysis to determine whether the on-treatment isolate was a genetically different strain compared with the pre-treatment isolate. RESULTS: Among 327 enrolled patients, we identified the on-treatment isolates of 198 patients as the same species as the pre-treatment isolates. The median treatment duration for the 198 patients was 14.4 months (interquartile range, 12.1-16.9 months). Of these patients, MLST analysis revealed the presence of a genetically different strain among the on-treatment isolates at least once in 24.7% (49/198) of patients (95% confidence interval, 18.9-31.4) compared to the pre-treatment isolate. There were variations in the timing, frequency, and number of distinct strains in these 49 patients. INTERPRETATION: We identified a genetically distinct strain within the same species at least once in approximately 25% of patients in whom the same species was isolated after the initiation of anti-MAC-PD therapy. These findings may affect the determination of treatment outcomes and corresponding MAC-PD treatment strategies.

Genotyping of Candida tropicalis isolates uncovers nosocomial transmission of two lineages in Italian tertiary-care hospital.

OBJECTIVES: Candida tropicalis is a medically important yeast with rising antifungal resistance, while nosocomial transmission is rarely reported. Here we genotyped C. tropicalis isolates from Italian hospitals to uncover potential nosocomial transmission and assess resistance. METHODS: A total of 197 C. tropicalis isolates from 161 patients was collected from five centres from 2013 to 2023. Short tandem repeat (STR) genotyping was conducted on all isolates and a selection of 24 isolates was typed with whole genome sequencing (WGS) and the novel Fourier-transform infrared (FTIR) spectroscopy method. Antifungal resistance was investigated with microbroth dilution and WGS. RESULTS: STR genotyping revealed seven clusters with isolates from multiple patients. WGS single nucleotide polymorphism (SNP) analysis on five groups of isolates with related STR genotypes also separated these isolates into five groups, of which two groups contained a cluster of isolates from different patients distinguished by 59 or fewer SNPs. In comparison, sequential isolates within three patients were differentiated by 141 SNPs at most. The two C. tropicalis WGS clusters also clustered based on FTIR genotyping, although this method did not separate the isolates into five groups. None of the 24 isolates were resistant to common antifungals. CONCLUSIONS: WGS SNP analysis indicated nosocomial transmission of two lineages within the same hospital, highlighting the need for enforced infection prevention measures and the need for routine genotyping on this common yeast in clinical settings. While both STR and FTIR genotyping also clustered these lineages, WGS SNP analysis is required to determine whether isolates were clonally transmitted.