Genotyping Strategy Research Articles

Predicted breeding values for relative scrapie susceptibility for genotyped and ungenotyped sheep

BackgroundScrapie is an infectious prion disease in sheep. Selective breeding for resistant genotypes of the prion protein gene (PRNP) is an effective way to prevent scrapie outbreaks. Genotyping all selection candidates in a population is expensive but existing pedigree records can help infer the probabilities of genotypes in relatives of genotyped animals.ResultsWe used linear models to predict allele content for the various PRNP alleles found in Icelandic sheep and compiled the available estimates of relative scrapie susceptibility (RSS) associated with PRNP genotypes from the literature. Using the predicted allele content and the genotypic RSS we calculated estimated breeding values (EBV) for RSS. We tested the predictions on simulated data under different scenarios that varied in the proportion of genotyped sheep, genotyping strategy, pedigree recording accuracy, genotyping error rates and assumed heritability of allele content. Prediction of allele content for rare alleles was less successful than for alleles with moderate frequencies. The accuracy of allele content and RSS EBV predictions was not affected by the assumed heritability, but the dispersion of prediction was affected. In a scenario where 40% of rams were genotyped and no errors in genotyping or recorded pedigree, the accuracy of RSS EBV for ungenotyped selection candidates was 0.49. If only 20% of rams were genotyped, or rams and ewes were genotyped randomly, or there were 10% pedigree errors, or there were 2% genotyping errors, the accuracy decreased by 0.07, 0.08, 0.03 and 0.04, respectively. With empirical data, the accuracy of RSS EBV for ungenotyped sheep was 0.46–0.65.ConclusionsA linear model for predicting allele content for the PRNP gene, combined with estimates of relative susceptibility associated with PRNP genotypes, can provide RSS EBV for scrapie resistance for ungenotyped selection candidates with accuracy up to 0.65. These RSS EBV can complement selection strategies based on PRNP genotypes, especially in populations where resistant genotypes are rare.

Retraction: M. Ashton, N. Czado, M. Harrel, S. Hughes. "Genotyping strategies for tissues fixed with various embalming fluids for human identification, databasing, and traceability," Journal of Forensic Sciences (Early View) https://doi.org/10.1111/1556-4029.15414.

Retraction: M. Ashton, N. Czado, M. Harrel, S. Hughes. "Genotyping strategies for tissues fixed with various embalming fluids for human identification, databasing, and traceability," Journal of Forensic Sciences (Early View) https://doi.org/10.1111/1556-4029.15414.

Phase IV adaptive randomised clinical trials evaluating efficacy and cost-efficacy of pre-emptive pharmacogenetic genotyping strategies in the Spanish National Health System: iPHARMGx Master Protocol and PREVESTATGx nested clinical trial

IntroductionGenetic variations impact drug response, driving the need for personalised medicine through pre-emptive pharmacogenetic testing. However, the adoption of pre-emptive pharmacogenetic testing for commonly prescribed drugs, such as statins, outside...

Real-world evaluation of CYP2C19 guided antiplatelet therapy in patients undergoing intracranial aneurysm repair.

Aim: To evaluate the feasibility and impact of using CYP2C19 genotype to guide selection of antiplatelet therapy in patients undergoing intracranial aneurysm treatment with a flow diversion stent in a real-world clinical setting.Patients & methods: A single-center, retrospective, observational cohort study was conducted in 112 patients undergoing intracranial aneurysm repair with flow-diversion stenting from 2014 to2021. Data were abstracted from health records. The frequency of clopidogrel or alternative therapy (ticagrelor or prasugrel) use was compared across CYP2C19 status (intermediate or poor metabolizer [IM/PM] vs. normal, rapid, or ultrarapid metabolizer [NM/RM/UM]).Results: In the study population, CYP2C19 genotype testing was performed on 110 (98.2%) patients; of these, 106 (97.2%) had results available prior to the stent procedure and 28 (25.5%) were IM/PMs. Alternative therapy was used more frequently in IM/PMs compared with NM/RM/UMs (57.1 vs. 8.5%, respectively, p<0.0001). The frequency of thromboembolic events over 12months did not significantly differ across clopidogrel-treated IM/PMs, clopidogrel-treated NM/RM/UMs and patients on alternative therapy (p=0.352); although, event numbers were low.Conclusion: A pre-emptive CYP2C19 genotyping strategy to guide antiplatelet therapy selection in intracranial aneurysm repair patients is feasible in a real-world clinical setting. Larger studies are needed to assess the impact on clinical outcomes.

Analysis of Antibiotic Response in Clinical Wound Pseudomonas aeruginosa Isolates: Unveiling Proteome Dynamics of Tobramycin-Tolerant Phenotype

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

Systematic Screening of Autosomal Dominant Tubulointerstitial Kidney Disease- MUC1 27dupC Pathogenic Variant through Exome Sequencing.

The MUC1 gene is associated with autosomal dominant tubulointerstitial kidney disease (ADTKD), leading to CKD. Current methods of sequencing, like exome sequencing, rarely detect MUC1 pathogenic variants because of the variable number of tandem repeats (VNTR) in MUC1 exon2. We demonstrated that combining fast read filtering with a sensitive VNTR genotyping strategy enables systematic screening of 27dupC pathogenic MUC1 variant from exome data. We initially validated our bioinformatics pipeline in a proof-of-concept cohort incorporating exome data from 33 participants with a known MUC1 pathogenic variant identified by Snapshot PCR and confirmed by 54 MUC1-negative individuals for negative control. We then retrospectively analyzed exome sequencing data from January 2019 to October 2023 from 3512 adult participants with nephropathy of unknown origin. Finally, we prospectively validated our pipeline in 825 additional participants enrolled from November 2023. SharkVNTyper accurately identified MUC1 variants in 32 out of 33 participants and excluded its presence in all the 54 negative controls in the proof-of-concept cohort (sensitivity of 97%, specificity of 100%). Integration of Shark tool with VNTyper significantly reduced running time from 6-12 hours to 5-10 minutes per sample, allowing both retrospective and prospective analysis. In the retrospective cohort, SharkVNTyper identified 23 additional positive cases that were not suspected clinically and had been missed in the initial exome analysis; 18 of these cases were confirmed as carrying the MUC1 27dupC mutation by low-throughput Snapshot PCR. In the prospective cohort of 825 CKD cases, systematic screening discovered 13 positive cases, with 12 confirmed by PCR. Overall, of 63 participants (1.4% of 4653) with molecularly confirmed ADTKD-MUC1, comprehensive diagnoses and descriptions of the disease were available for 24 participants. Median age of kidney failure was 50 years, 38% exhibited bilateral multiple kidney cysts, 8% had early-onset gout, and 58% had arterial hypertension. SharkVNTyper enables the analysis of highly repeated regions, such as the MUC1 VNTR, and facilitates the systematic screening of ADTKD-MUC1 from exome data, fostering 27dupC variation identification.

Genotyping both live and dead animals to improve post-weaning survival of pigs in breeding programs

BackgroundIn this study, we tested whether genotyping both live and dead animals (GSD) realises more genetic gain for post-weaning survival (PWS) in pigs compared to genotyping only live animals (GOS).MethodsStochastic simulation was used to estimate the rate of genetic gain realised by GSD and GOS at a 0.01 rate of pedigree-based inbreeding in three breeding schemes, which differed in PWS (95%, 90% and 50%) and litter size (6 and 10). Pedigree-based selection was conducted as a point of reference. Variance components were estimated and then estimated breeding values (EBV) were obtained in each breeding scheme using a linear or a threshold model. Selection was for a single trait, i.e. PWS with a heritability of 0.02 on the observed scale. The trait was simulated on the underlying scale and was recorded as binary (0/1). Selection candidates were genotyped and phenotyped before selection, with only live candidates eligible for selection. Genotyping strategies differed in the proportion of live and dead animals genotyped, but the phenotypes of all animals were used for predicting EBV of the selection candidates.ResultsBased on a 0.01 rate of pedigree-based inbreeding, GSD realised 14 to 33% more genetic gain than GOS for all breeding schemes depending on PWS and litter size. GSD increased the prediction accuracy of EBV for PWS by at least 14% compared to GOS. The use of a linear versus a threshold model did not have an impact on genetic gain for PWS regardless of the genotyping strategy and the bias of the EBV did not differ significantly among genotyping strategies.ConclusionsGenotyping both dead and live animals was more informative than genotyping only live animals to predict the EBV for PWS of selection candidates, but with marginal increases in genetic gain when the proportion of dead animals genotyped was 60% or greater. Therefore, it would be worthwhile to use genomic information on both live and more than 20% dead animals to compute EBV for the genetic improvement of PWS under the assumption that dead animals reflect increased liability on the underlying scale.

Genomic selection strategies for the German Merino sheep breeding programme - A simulation study.

Genomic selection is widely implemented in livestock breeding programmes across species. Its potential is also evident for sheep breeding; however, it has several limitations, particularly because of the high genetic diversity across and within sheep breeds. In Germany, the predominant sheep breed is the Merino sheep. Until now, there has been no use of genomic selection in the German Merino sheep breeding programme. In this simulation study, different genomic selection strategies were compared with a reference scenario with a breeding value estimation based on pedigree BLUP. A simplified version of the German Merino sheep breeding programme, including a health and a production trait in the breeding goal, was simulated via the R package Modular Breeding Program Simulator (MoBPS). Real genotype data were used to create a population specific simulation. The reference scenario was compared with several alternative scenarios in which selection was based on single-step GBLUP (ssGBLUP) breeding value estimation with varying genotyping strategies. In addition to scenarios in which all male and all male plus all female lambs were genotyped, scenarios with a preselection of lambs, that is only a certain proportion (top 25%, top 50%) genotyped, were simulated. The results revealed that genetic gain increased with increasing numbers of available genotypes. However, marginal gains decreased with increasing numbers of genotypes. Compared with the reference scenario, genotyping the top 25% of male lambs increased the genetic gain for the breeding ram population by 13% for both traits whereas genotyping the top 50% of male lambs or all male lambs led to increases of 18% (17%) or 26% (21%) for the health (production) trait, respectively. The potential of genotyping females in addition to male lambs was less evident on the male side with no significant differences between the scenarios with different proportions of genotyped females. The results have shown that genomic selection can be a valuable tool to increase genetic gain in the German Merino sheep population and that the genotyping of a certain proportion of animals might lead to substantial improvement over pedigree-based breeding value estimation. Nevertheless, further studies, especially economic evaluations, are needed before practical implementation.

Development of an efficient molecular-marker assisted selection strategy for berry color in grapevine

Marker-assisted selection (MAS) represents one of the most effective strategies to reduce costs and time in plant breeding programs. MAS benefits from the identification of markers in strong linkage disequilibrium (LD) with causative genes and from the development of fast, scalable, and reproducible screening techniques. In grape, berry color is a valuable trait, highly related to the sensory characteristics of fresh grape and wine quality.The present work aims to develop a MAS strategy to select skin color by High-Resolution Melting (HRM), a ‘close-tube’ method capable of detecting polymorphisms within PCR-amplified DNA fragments. To identify molecular markers associated with the berry skin color phenotypic trait, the total genome of 3 white-berry and 3 red-berry accessions were sequenced using Illumina technology and aligned to the reference genome PN40024 v4. The sequencing results revealed the presence of three highly polymorphic regions on chromosome 2 between 15 Kbp and 17 Kbp for red berry genotypes. HRM analyses were carried out on a total of 95 genotypes, 70 of which comprised a berry color segregating population: [‘Italia’ (white berry) x cv ‘Crimson Seedless’ (red berry)] and 25 genotypes belonging to a commercial grapevine germplasm collection consisting of 11 red and 14 white berry accessions. The HRM profiles of the three regions tested strongly correlated with the phenotypic trait of color.The results of this work represent an effective, fast, and reliable genotyping strategy that enables the discrimination of genotypes based on the presence or absence of berry pigmentation.

Phenotypic spectrum in Weiss-Kruszka syndrome caused by ZNF462 variants: Three new patients and literature review

Weiss-Kruszka Syndrome (WSKA) is caused by pathogenic variants in ZNF462 representing a rare autosomal dominant congenital anomaly syndrome. It is characterized by global developmental delay, hypotonia, feeding difficulties, and craniofacial abnormalities, documented in fewer than 30 patients. ZNF462, located on chromosome 9p31.2, is a transcription factor and has an important role during embryonic development and chromatin remodelling. Here, we report three new patients with WSKA, Through whole exome sequencing (WES) analysis, we identified two novel variants in three patients, two of whom are siblings. These variants (c.3078dup, p.Val1027Cysfs5 and c.4792A > T p.Lys1598*) in the ZNF462 gene are likely resulting in haploinsufficiency. Our patients help to further delineate the phenotype, genotype and potential therapeutic management strategies for WSKA. Since we report a second WSKA patient with an autoimmune disease further clinical and functional studies are needed to elucidate the association between this chromatin remodelling disorder and the development of autoimmune problems. In the future, collaborative efforts are encouraged to develop an episignature for WSKA, given the gene's function and associated patient phenotypes. This new technology has the potential to provide valuable insights into the disorder.

Identification of distinct genotypes in circulating RSV A strains based on variants in the virus replication-associated genes.

Respiratory syncytial virus (RSV) is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroups, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the United States. In-depth analysis and annotation of variants in the replication-associated proteins identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its co-factor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. Similar associations were seen among sequences of the related human metapneumovirus. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates.IMPORTANCEGiven the historical heterogeneity of respiratory syncytial virus (RSV) and the disease it causes, there is a need to understand the properties of the circulating RSV strains each season. This information would benefit from an informative and consensus method of genotyping the virus. Here, we carried out a variant analysis that shows a pattern of specific variations among the replication-associated genes of RSV A across different seasons. Interestingly, these variation patterns, which were also seen in human metapneumovirus sequences, point to previously defined interactions of domains within these genes, suggesting co-variation in the replication-associated genes. Our results also suggest a genotyping strategy that can prove to be particularly important in understanding the genotype-phenotype correlation in the era of RSV vaccination, where selective pressure on the virus to evolve is anticipated. More importantly, the categorization of pneumoviruses based on these patterns may be of prognostic value.

Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme

ObjectivesWe propose fast and accurate molecular detection of the Y132F ERG11p substitution directly on pure-cultured Candida parapsilosis isolates. We also assessed a discriminative genotyping scheme to track circulating genotypes. MethodsA total of 223 C. parapsilosis isolates (one patient each) from 20 hospitals, located in Spain and Italy were selected. Isolates were fluconazole-resistant (n = 94; harbouring the Y132F ERG11p substitution [n = 85], the G458S substitution [n = 6], the R398I substitution [n = 2], or the wild-type ERG11 gene sequence) or fluconazole-susceptible (n = 129). Two targeted-A395T-mutation PCR formats (conventional and real-time) were engineered and optimized on fluconazole-susceptible and fluconazole-resistant pure-cultured isolates, thus skipping DNA extraction. Two genotyping schemes were compared: Scheme 1 (CP1, CP4a, CP6, and B markers), and Scheme 2 (6A, 6B, 6C, CP1, CP4a, and CP6 markers). ResultsThe screening performed using both PCR formats showed 100% specificity (fluconazole-susceptible isolates; n = 129/129) and sensitivity (Y132F isolates; n = 85/85) values; however, results were available in 3 and 1.5 hours with the conventional and real-time PCR formats, respectively. Overall, Scheme 1 showed higher genetic diversity than Scheme 2, as shown by the number of alleles detected (n = 98; mean 23, range 13–38), the significantly higher observed and expected heterozygosity, and the probability of identity index (2.5 × 10−6). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes. ConclusionBoth proposed PCR formats allow us to speed up the accurate detection of substitution Y132F ERG11p in C. parapsilosis isolates with 100% specificity and sensitivity. In addition, we recommend CP1, CP4a, CP6, and B microsatellite markers for genotyping fluconazole-resistant isolates.

Tagging large CNV blocks in wheat boosts digitalization of germplasm resources by ultra-low-coverage sequencing

BackgroundThe massive structural variations and frequent introgression highly contribute to the genetic diversity of wheat, while the huge and complex genome of polyploid wheat hinders efficient genotyping of abundant varieties towards accurate identification, management, and exploitation of germplasm resources.ResultsWe develop a novel workflow that identifies 1240 high-quality large copy number variation blocks (CNVb) in wheat at the pan-genome level, demonstrating that CNVb can serve as an ideal DNA fingerprinting marker for discriminating massive varieties, with the accuracy validated by PCR assay. We then construct a digitalized genotyping CNVb map across 1599 global wheat accessions. Key CNVb markers are linked with trait-associated introgressions, such as the 1RS·1BL translocation and 2NvS translocation, and the beneficial alleles, such as the end-use quality allele Glu-D1d (Dx5 + Dy10) and the semi-dwarf r-e-z allele. Furthermore, we demonstrate that these tagged CNVb markers promote a stable and cost-effective strategy for evaluating wheat germplasm resources with ultra-low-coverage sequencing data, competing with SNP array for applications such as evaluating new varieties, efficient management of collections in gene banks, and describing wheat germplasm resources in a digitalized manner. We also develop a user-friendly interactive platform, WheatCNVb (http://wheat.cau.edu.cn/WheatCNVb/), for exploring the CNVb profiles over ever-increasing wheat accessions, and also propose a QR-code-like representation of individual digital CNVb fingerprint. This platform also allows uploading new CNVb profiles for comparison with stored varieties.ConclusionsThe CNVb-based approach provides a low-cost and high-throughput genotyping strategy for enabling digitalized wheat germplasm management and modern breeding with precise and practical decision-making.

A genome-wide association study and an accurate genotyping strategy to identify and validate the SNPs related to the larval period in bullfrog Aquarana catesbeiana

A genome-wide association study and an accurate genotyping strategy to identify and validate the SNPs related to the larval period in bullfrog Aquarana catesbeiana

Molecular detection and genetic characteristics of porcine circovirus 3 and porcine circovirus 4 in central China.

Porcine circoviruses (PCVs) are a significant cause of concern for swine health, with four genotypes currently recognized. Two of these, PCV3 and PCV4, have been detected in pigs across all age groups, in both healthy and diseased animals. These viruses have been associated with various clinical manifestations, including porcine dermatitis and nephropathy syndrome (PDNS) and respiratory and enteric signs. In this study, we detected PCV3 and PCV4 in central China between January 2022 and February 2023. We tested fecal swabs and tissue samples from growing-finishing and suckling pigs with or without respiratory and systemic manifestations and found the prevalence of PCV3 to be 15.15% (15/99) and that of PCV3/PCV4 coinfection to be 4.04% (4/99). This relatively low prevalence might be attributed to the fact that most of the clinical samples were collected from pigs exhibiting respiratory signs, with only a few samples having been obtained from pigs with diarrhea. In some cases, PCV2 was also detected, and the coinfection rates of PCV2/3, PCV2/4, and PCV2/3/4 were 6.06% (6/99), 5.05% (5/99), and 3.03% (3/99), respectively. The complete genomic sequences of four PCV3 and two PCV4 isolates were determined. All four of the PCV3 isolates were of subtype PCV3b, and the two PCV4 isolates were of subtype PCV4b. Two mutations (A24V and R27K) were found in antibody recognition domains of PCV3, suggesting that they might be associated with immune escape. This study provides valuable insights into the molecular epidemiology and evolution of PCV3 and PCV4 that will be useful in future investigations of genotyping, immunogenicity, and immune evasion strategies.

Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern

Giardia duodenalis is one of the most common causes of waterborne disease worldwide, and is often associated with outbreaks of diarrhea in areas with poor sanitation and hygiene. This study aimed to assess the prevalence and genetic diversity of G. duodenalis assemblages in individuals attending major public hospitals in Shiraz, southwestern Iran. From August 2022 to May 2023, a total of 614 stool samples from individuals were collected and initially examined for G. duodenalis cysts using parasitological techniques, sucrose flotation, and microscopy. Microscopy-positive samples were validated by SSU-PCR amplification of the parasite DNA. A multilocus genotyping (MLG) scheme, which focused on the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes, was employed for genotyping purposes. G. duodenalis cysts were found in 7.5% (46/614) and 8.5% (52/614) of samples through microscopy and SSU-PCR, respectively. Successful amplification and sequencing results were obtained for 77.3% (17/22) and 45.5% (10/22) of the infected samples at the tpi and gdh loci, respectively. MLG data for the two loci were available for only five samples. Out of the 22 samples genotyped at any loci, 54.5% (12/22) were identified as assemblage A, while 45.5% (10/22) were identified as assemblage B. AII was the most predominant sub-assemblage identified [54.5% (12/22)], followed by BIII [27% (6/22)], discordant BIII/BIV [13.6% (3/22)], and BIV [4.5% (1/22)]. In the present study, no assemblages suited for non-human animal hosts (e.g., CF) were detected. This suggests that the transmission of human giardiasis in Shiraz is primarily anthroponotic. Further molecular-based analyses are necessary to confirm and expand upon these findings. These analyses will also help determine the presence and public health importance of the parasite in environmental samples, such as drinking water.

Colposcopy Referral and CIN3+ Risk of Human Papillomavirus Genotyping Strategies in Cervical Cancer Screening.

High-risk human papillomavirus (hrHPV)-based cervical cancer screening in the Netherlands led to a substantial increase in number of colposcopy referrals and low-grade lesions detected. Genotyping strategies may be employed to lower the screening-related burden. We evaluated 14 triage strategies with genotyping (HPV16/18 or HPV16/18/31/33/45/52/58) for hrHPV-positive borderlineormilddyskaryosis (BMD)ornormal cytology,usingdata from a population-based hrHPV-based screening trial with 5-year interval (POBASCAM). We considered colposcopy referral at baseline, after 6-month repeat cytology and after 5-year hrHPV testing. Performance was evaluated by one-round positive and negative predictive value (PPVandNPV) for CIN3+ and by two-roundcolposcopy referral rate. To identify efficient strategies, they were ordered by the one-round colposcopy referral rate. Adjacent strategies were compared by the marginal PPV for detecting one additional CIN3+ (mPPV). The most conservative strategy (repeat cytology after BMD and HPV16/18/31/33/45/52/58-positive normal cytology, next round otherwise) yielded an mPPV of 28%, NPV of 98.2%, and two-round colposcopy referral rate of 47.2%. Adding direct referral after BMD or genotype-positive BMD yielded an mPPV ≤ 8.2%, NPV ≥ 98.5% and an increase in colposcopy referral rate of 1.9% to 6.5%. Adding direct referral after HPV16/18-positive normal cytology yielded an mPPV ≤ 3.5%, NPV ≥ 99.5% and an increase in colposcopy referral rate of 13.9%. Direct colposcopy referral of women with BMD or normal cytology is unlikely to be efficient, but genotype-guided direct referral after BMD may be considered because the increase in colposcopies is limited. hrHPV screening programs can become very efficient when immediate colposcopy referral is limited to women at highest CIN3+ risk. See related In the Spotlight, p. 979.

Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Dynamic Patterns and Predominance of Respiratory Pathogens Post-COVID-19: Insights from a Two-Year Analysis

IntroductionRespiratory tract infections (RTIs) stand out as the most frequent causes leading to visits to the emergency department and hospitalizations. This study aims to assess the types and prevalence of respiratory infections across two years following the end of the COVID-19 pandemic.MethodsPatients presenting with an influenza-like illness (ILI) were tested using multiplex RT-PCR (QIAstat-Dx, Qiagen). The multiplexed RT- PCR test detects 21 respiratory viruses and bacteria.ResultsDuring the study period, PCR test was done on a total of 1,790 samples were tested, and 712 (40%) were positive for a total of 796 pathogens. The mean age (± SD) of the participants was 20.1 ± 28.4 years in 2022 and 21.9 ± 27.6 years in 2023. Among the detected pathogens, the most prevalent were Rhinovirus/Enterovirus 222 (12.4%), followed by RSV A&B (103 cases, 5.7%), and H1N1 Influenza (77 cases, 4.3%). Additionally, Influenza A/B constituted 172 (9.6%) while parainfluenza constituted (58, 3.2%). SARS-CoV-2 was identified in 3.97% of the samples. Over the two-year period, the monthly pattern of the identified pathogens exhibited fluctuations in the prevalence. Furthermore, variations were observed in the detected pathogens across different age groups.ConclusionIn addition to adding significant knowledge to the field of respiratory viral infections, this study emphasizes the necessity of ongoing research and surveillance for the detection and characterization of respiratory viruses, particularly those with the potential for emergence. Such studies would also require setting up a strategy for genotyping and/or sequencing of viruses.

Novel typing scheme reveals emergence and genetic diversity of Chlamydia pecorum at the local management scale across two koala populations

To overcome shortcomings in discriminating Chlamydia pecorum strains infecting the koala (Phascolarctos cinereus) at the local level, we developed a novel genotyping scheme for this pathogen to inform koala management at a fine-scale subpopulation level. We applied this scheme to two geographically distinct koala populations in New South Wales, Australia: the Liverpool Plains and the Southern Highlands to South-west Sydney (SHSWS). Our method provides greater resolution than traditional multi-locus sequence typing, and can be used to monitor strain emergence, movement, and divergence across a range of fragmented habitats. Within the Liverpool Plains population, suspected recent introduction of a novel strain was confirmed by an absence of genetic diversity at the earliest sampling events and limited diversity at recent sampling events. Across the partially fragmented agricultural landscape of the Liverpool Plains, diversity within a widespread sequence type suggests that this degree of fragmentation may hinder but not prevent spread. In the SHSWS population, our results suggest movement of a strain from the south, where diverse strains exist, into a previously Chlamydia-free area in the north, indicating the risk of expansion towards an adjacent Chlamydia-negative koala population in South-west Sydney. In the south of the SHSWS where koala subpopulations appear segregated, we found evidence of divergent strain evolution. Our tool can be used to infer the risks of strain introduction across fragmented habitats in population management, particularly through practices such as wildlife corridor constructions and translocations.