A healthy live birth after mosaic blastocyst transfer in preimplantation genetic testing for GATA1-related cytopenia combined with HLA matching.

BACKGROUND: GATA1-related cytopenia (GRC) is characterized by thrombocytopaenia and/or anaemia ranging from mild to severe. Haematopoietic stem cell transplantation (HSCT) is a healing therapeutic choice for GRC patients. We identified a novel pathogenic variant (GATA1: c.1019delG) in a boy with GATA1-related cytopenia. Then we performed preimplantation genetic testing (PGT) in this GRC family. After a mosaic embryo transfered, a healthy and HLA-compatible with the proband baby was delivered. CASE PRESENTATION: The proband is a 6-year-old boy who was diagnosed to have transfusion-dependent anaemia since 3 year old. Whole-exome sequencing (WES) showed that the proband has a hemizygous variant c.1019delG in GATA1, which is inherited from his mother. His parents decided to undergo PGT to have a health and HLA-compatible offspring. After whole genome amplification (WGA) of biopsied trophectoderm (TE) cells, next generation sequencing (NGS)-based PGT was preformed to analyse embryos on chromosomal aneuploidy, target mutation and HLA typing. There were 3 embryos HLA-matched to the proband. The genotypes of the 3 embryos were heterozygous variant, hemizygous variant, normal respectively. After a heterozygous, mosaic partial trisomy (chr)16, and HLA-matched embryo transfer, a healthy baby was delivered and whose HSCT is compatible with the proband. CONCLUSIONS: NGS-based PGT-HLA is a valuable procedure for the treatment of GATA1-related cytopenia caused by GATA1 variants, or other haematological disorders, oncological and immunological diseases. Furthermore, our study reconfirms that mosaic embryos transfer would bring healthy offspring.

Genetic specialization of key bifidobacterial phylotypes in multiple mother-infant dyad cohorts from geographically isolated populations.

Little has been known about symbiotic relationships and host specificity for symbionts in the human gut microbiome so far. Bifidobacteria are a paragon of the symbiotic bacteria biota in the human gut. In this study, we characterized the population genetic structure of three bifidobacteria species from 58 healthy mother-infant pairs of three ethnic groups in China, geographically isolated, by Rep-PCR, multi-locus sequence analysis (MLSA), and in vitro carbohydrate utilization. One hundred strains tested were incorporated into 50 sequence types (STs), of which 29 STs, 17 STs, and 4 STs belong to B. longum subsp. longum, B. breve, and B. animalis subsp. lactis, respectively. The conspecific strains from the same mother-child pair were genetically very similar, supporting the vertical transmission of Bifidobacterium phylotypes from mother to offspring. In particular, results based on allele profiles and phylogeny showed that B. longum subsp. longum and B. breve exhibited considerable intraspecies genetic heterogeneity across three ethnic groups, and strains were clustered into ethnicity-specific lineages. Yet almost all strains of B. animalis subsp. lactis were incorporated into the same phylogenetic clade, regardless of ethnic origin. Our findings support the hypothesis of co-evolution between human gut symbionts and their respective populations, which is closely linked to the lifestyle of specific bacterial lineages. Hence, the natural and evolutionary history of Bifidobacterium species would be an additional consideration when selecting bifidobacterial strains for industrial and therapeutic applications.

Cross-talk of MLST and transcriptome unveiling antibiotic resistance mechanism of carbapenem resistance Acinetobacter baumannii clinical strains isolated in Guiyang, China.

Introduction: Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing nosocomial infection in the clinic. The occurrence rate of antibiotic resistance is increasing year by year, resulting in a highly serious situation of bacterial resistance. Methods: To better understand the local epidemiology of multidrug-resistant A. baumannii, an investigation was conducted on the antibiotic resistance of different types of A. baumannii and its relationship with the genes of A. baumannii. Furthermore, the molecular mechanism underlying antibiotic resistance in A. baumannii was investigated through transcriptome analysis. Results: These results showed that a total of 9 STs were detected. It was found that 99% of the strains isolated in the hospital belonged to the same STs, and the clone complex CC208 was widely distributed in various departments and all kinds of samples. Furthermore, these A. baumannii strains showed high resistance to ertapenem, biapenem, meropenem, and imipenem, among which the resistance to ertapenem was the strongest. The detection rate of bla OXA-51 gene in these carbapenem resistance A. baumannii (CRAB) reached 100%; Additionally, the transcriptome results showed that the resistance genes were up-regulated in resistance strains, and these genes involved in biofilm formation, efflux pumps, peptidoglycan biosynthesis, and chaperonin synthesis. Discussion: These results suggest that the CC208 STs were the main clonal complex, and showed high carbapenem antibiotic resistance. All these resistant strains were distributed in various departments, but most of them were distributed in intensive care units (ICU). The bla OXA-23 was the main antibiotic resistance genotype; In summary, the epidemic trend of clinical A. baumannii in Guiyang, China was analyzed from the molecular level, and the resistance mechanism of A. baumannii to carbapenem antibiotics was analyzed with transcriptome, which provided a theoretical basis for better control of A. baumannii.

Application of Pathogen Genomics to Outbreak Investigation.

Outbreaks are a risk to public health particularly when pathogenic, hypervirulent, and/or multidrug-resistant organisms (MDROs) are involved. In a hospital setting, vulnerable populations such as the immunosuppressed, intensive care patients, and neonates are most at risk. Rapid and accurate outbreak detection is essential to implement effective interventions in clinical areas to control and stop further transmission. Advances in the field of whole genome sequencing (WGS) have resulted in lowered costs, increased capacity, and improved reproducibility of results. WGS now has the potential to revolutionize the investigation and management of outbreaks replacing conventional genotyping and other discrimination systems. Here, we outline specific procedures and protocols to implement WGS into investigation of outbreaks in healthcare settings.

Use of ultrasound imaging Omics in predicting molecular typing and assessing the risk of postoperative recurrence in breast cancer.

BACKGROUND: The aim of this study is to assess the efficacy of a multiparametric ultrasound imaging omics model in predicting the risk of postoperative recurrence and molecular typing of breast cancer. METHODS: A retrospective analysis was conducted on 534 female patients diagnosed with breast cancer through preoperative ultrasonography and pathology, from January 2018 to June 2023 at the Affiliated Cancer Hospital of Xinjiang Medical University. Univariate analysis and multifactorial logistic regression modeling were used to identify independent risk factors associated with clinical characteristics. The PyRadiomics package was used to delineate the region of interest in selected ultrasound images and extract radiomic features. Subsequently, radiomic scores were established through Least Absolute Shrinkage and Selection Operator (LASSO) regression and Support Vector Machine (SVM) methods. The predictive performance of the model was assessed using the receiver operating characteristic (ROC) curve, and the area under the curve (AUC) was calculated. Evaluation of diagnostic efficacy and clinical practicability was conducted through calibration curves and decision curves. RESULTS: In the training set, the AUC values for the postoperative recurrence risk prediction model were 0.9489, and for the validation set, they were 0.8491. Regarding the molecular typing prediction model, the AUC values in the training set and validation set were 0.93 and 0.92 for the HER-2 overexpression phenotype, 0.94 and 0.74 for the TNBC phenotype, 1.00 and 0.97 for the luminal A phenotype, and 1.00 and 0.89 for the luminal B phenotype, respectively. Based on a comprehensive analysis of calibration and decision curves, it was established that the model exhibits strong predictive performance and clinical practicability. CONCLUSION: The use of multiparametric ultrasound imaging omics proves to be of significant value in predicting both the risk of postoperative recurrence and molecular typing in breast cancer. This non-invasive approach offers crucial guidance for the diagnosis and treatment of the condition.

Spatial Deconvolution of Cell Types and Cell States at Scale Utilizing TACIT.

Identifying cell types and states remains a time-consuming, error-prone challenge for spatial biology. While deep learning is increasingly used, it is difficult to generalize due to variability at the level of cells, neighborhoods, and niches in health and disease. To address this, we developed TACIT, an unsupervised algorithm for cell annotation using predefined signatures that operates without training data. TACIT uses unbiased thresholding to distinguish positive cells from background, focusing on relevant markers to identify ambiguous cells in multiomic assays. Using five datasets (5,000,000-cells; 51-cell types) from three niches (brain, intestine, gland), TACIT outperformed existing unsupervised methods in accuracy and scalability. Integrating TACIT-identified cell types with a novel Shiny app revealed new phenotypes in two inflammatory gland diseases. Finally, using combined spatial transcriptomics and proteomics, we discovered under- and overrepresented immune cell types and states in regions of interest, suggesting multimodality is essential for translating spatial biology to clinical applications.

Prevalence, Antimicrobial Resistance, and Diversity of Campylobacter Isolated from U.S. Goat Feces: 2019 NAHMS Survey.

Goats are often asymptomatic carriers of Campylobacter, including the foodborne pathogen Campylobacter jejuni. Infections can have significant and economically detrimental health outcomes in both humans and animals. The primary objective of this study was to estimate the prevalence of Campylobacter in U.S. goat herds. Campylobacter species were isolated from 106 of 3,959 individual animals and from 42 of 277 goat operations that participated in fecal sample collection as part of the National Animal Health Monitoring System Goat 2019 study. Weighted animal-level prevalence was 2.3% (SE = 0.5%) and operation prevalence was 13.0% (SE = 3.2%). Animal-level prevalence ranged widely from 0 to 70.0%, however, 52.4% of positive operations (22/42) had only a single isolate. C. jejuni was the most frequently isolated species (68.9%; 73/106), followed by C. coli (29.3%, 31/106). A total of 46.2% (36/78) of viable isolates were pan-susceptible to 8 antimicrobials. Resistance to tetracycline (TET) was observed in 44.9% (35/78) of isolates, while 12.8% (10/78) were resistant to ciprofloxacin (CIP) and nalidixic acid (NAL). Among all isolates, a single resistance profile CIP-NAL-TET was observed in 3.8% (3/78) of isolates. A total of 35 unique sequence types (STs) were identified, 11 of which are potentially new. Multiple C. jejuni STs were observed in 48.1% (13/27) of positive operations. Goats with access to surface water, operations reporting antibiotics in the feed or water (excluding ionophores and coccidiostats), and operations reporting abortions and without postabortion management tasks had significantly greater odds of being Campylobacter positive. This snapshot of the U.S. goat population enriches the limited pool of knowledge on Campylobacter species presence in U.S. goats.

Serotype distribution, antibiotic resistance, multilocus sequence typing, and virulence factors of invasive and non-invasive Streptococcus pneumoniae in Northeast China from 2000 to 2021.

Streptococcus pneumoniae infection is a major public health concern with high morbidity and mortality rates. This study aimed to evaluate the serotype distribution, antimicrobial resistance changes, clonal composition, and virulence factors of S. pneumoniae isolates causing pneumococcal disease in northeast China from 2000 to 2021. A total of 1,454 S. pneumoniae isolates were included, with 568 invasive strains and 886 non-invasive strains. The patients from whom the S. pneumoniae were isolated ranged in age from 26 days to 95 years, with those ≤ 5 years old comprising the largest group (67.19%). 19 F, 19 A, 23 F, 14, and 6B were the most common serotypes, of which 19 A and 19 F were the main serotypes of invasive and non-invasive S. pneumoniae, respectively. CC271 was the most common multilocus sequence type. Serotype 14 had the lowest expression of cbpA, rrgA, and psrP genes, but expression levels of 19 A and 19 F genes were similar. All isolates were sensitive to ertapenem, moxifloxacin, linezolid, and vancomycin but highly resistant to macrolides, tetracyclines, and cotrimoxazole. Simultaneous resistance to erythromycin, clindamycin, tetracyclines, and trimethoprim/sulfamethoxazole was common pattern among multidrug-resistant isolates. Non-invasive S. pneumoniae had higher resistance to ß-lactam antibiotics than invasive strains. 19 A and 19 F were the main strains of penicillin-resistant S. pneumoniae. The resistance rate of ß-lactam antibiotics decreased from 2017 to 2021 compared to previous periods. Including PCV13 in the national immunization program can reduce the morbidity and mortality rates of pneumococcal disease effectively.

Nosocomial transmission of tet(x3), bla NDM-1 and bla OXA-97-carrying Acinetobacter baumannii conferring resistance to eravacycline and omadacycline, the Netherlands, March to August 2021.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.

Orthanq: transparent and uncertainty-aware haplotype quantification with application in HLA-typing.

BACKGROUND: Identification of human leukocyte antigen (HLA) types from DNA-sequenced human samples is important in organ transplantation and cancer immunotherapy and remains a challenging task considering sequence homology and extreme polymorphism of HLA genes. RESULTS: We present Orthanq, a novel statistical model and corresponding application for transparent and uncertainty-aware quantification of haplotypes. We utilize our approach to perform HLA typing while, for the first time, reporting uncertainty of predictions and transparently observing mutations beyond reported HLA types. Using 99 gold standard samples from 1000 Genomes, Illumina Platinum Genomes and Genome In a Bottle projects, we show that Orthanq can provide overall superior accuracy and shorter runtimes than state-of-the-art HLA typers. CONCLUSIONS: Orthanq is the first approach that allows to directly utilize existing pangenome alignments and type all HLA loci. Moreover, it can be generalized for usages beyond HLA typing, e.g. for virus lineage quantification. Orthanq is available under https://orthanq.github.io .

Identification of the novel HLA-A*30:221 allele by next-generation sequencing.

The novel HLA-A*30:221 allele differs from HLA-A*30:01:01:01 by one nucleotide substitution in Exon 7.

Unveiling the microevolution of antimicrobial resistance in selected Pseudomonas aeruginosa isolates from Egyptian healthcare settings: A genomic approach.

The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance.

Smartphone-Based Assessment of Mobility and Manual Dexterity in Adult People with Spinal Muscular Atrophy.

Background: More responsive, reliable, and clinically valid endpoints of disability are essential to reduce size, duration, and burden of clinical trials in adult persons with spinal muscular atrophy (aPwSMA). Objective: The aim is to investigate the feasibility of smartphone-based assessments in aPwSMA and provide evidence on the reliability and construct validity of sensor-derived measures (SDMs) of mobility and manual dexterity collected remotely in aPwSMA. Methods: Data were collected from 59 aPwSMA (23 walkers, 20 sitters and 16 non-sitters) and 30 age-matched healthy controls (HC). SDMs were extracted from five smartphone-based tests capturing mobility and manual dexterity, which were administered in-clinic and remotely in daily life for four weeks. Reliability (Intraclass Correlation Coefficients, ICC) and construct validity (ability to discriminate between HC and aPwSMA and correlations with Revised Upper Limb Module, RULM and Hammersmith Functional Scale - Expanded HFMSE) were quantified for all SDMs. Results: The smartphone-based assessments proved feasible, with 92.1% average adherence in aPwSMA. The SDMs allowed to reliably assess both mobility and dexterity (ICC > 0.75 for 15/22 SDMs). Twenty-one out of 22 SDMs significantly discriminated between HC and aPwSMA. The highest correlations with the RULM were observed for SDMs from the manual dexterity tests in both non-sitters (Typing, ρ= 0.78) and sitters (Pinching, ρ= 0.75). In walkers, the highest correlation was between mobility tests and HFMSE (5 U-Turns, ρ= 0.79). Conclusions: This exploratory study provides preliminary evidence for the usability of smartphone-based assessments of mobility and manual dexterity in aPwSMA when deployed remotely in participants' daily life. Reliability and construct validity of SDMs remotely collected in real-life was demonstrated, which is a pre-requisite for their use in longitudinal trials. Additionally, three novel smartphone-based performance outcome assessments were successfully established for aPwSMA. Upon further validation of responsiveness to interventions, this technology holds potential to increase the efficiency of clinical trials in aPwSMA.

Development and implementation of a core genome multilocus sequence typing scheme for Yersinia enterocolitica: a tool for surveillance and outbreak detection.

Yersinia enterocolitica (Y. enterocolitica) is the most frequent etiological agent of yersiniosis and has been responsible for several national outbreaks in Norway and elsewhere. A standardized high-resolution method, such as core genome Multilocus Sequence Typing (cgMLST), is needed for pathogen traceability at the national and international levels. In this study, we developed and implemented a cgMLST scheme for Y. enterocolitica. We designed a cgMLST scheme in SeqSphere + using high-quality genomes from different Y. enterocolitica biotype sublineages. The scheme was validated if more than 95% of targets were found across all tested Y. enterocolitica: 563 Norwegian genomes collected between 2012 and 2022 and 327 genomes from public data sets. We applied the scheme to known outbreaks to establish a threshold for identifying major complex types (CTs) based on the number of allelic differences. The final cgMLST scheme included 2,582 genes with a median of 97.9% (interquartile range 97.6%-98.8%) targets found across all tested genomes. Analysis of outbreaks identified all outbreak strains using single linkage clustering at four allelic differences. This threshold identified 311 unique CTs in Norway, of which CT18, CT12, and CT5 were identified as the most frequently associated with outbreaks. The cgMLST scheme showed a very good performance in typing Y. enterocolitica using diverse data sources and was able to identify outbreak clusters. We recommend the implementation of this scheme nationally and internationally to facilitate Y. enterocolitica surveillance and improve outbreak response in national and cross-border outbreaks.

Genetic Diversity and Phylogenetic Study of Leishmania Species in Iran by Multilocus Sequence Typing.

Background: Leishmaniasis is an important public health parasitic infection, which is endemic in many parts of the world, including Iran. We aimed to investigate genetic diversity and phylogenetic relationship among different Leishmania isolates using multi-locus sequence typing (MLST). Methods: Totally, 41 isolates collected either from patients referred to Leishmaniasis Diagnostics and Treatment Center at Tehran University of Medical Sciences, Tehran, Iran or from animals during 2019-2021, were subjected to the study. They included L. major and L. tropica from human, L. infantum from canine, and L. turanica from rodents from different endemic foci of Iran analyzed using MLST including gp63, g6pdh, lack, nagt, and hsp70 genes. Results: A total of 5010 bps was analyzed from each isolate. The three targets, nagt, lack, and g6pdh, generated better topology comparing to the other genes. In the 44 isolates, 22 haplotypes (STs) were identified. Leishmania tropica contained the highest number of haplotypes (n=12) comparing to L. major (n=8), L. infantum (n=1) and L. turanica (n=1). All five genomic loci caused separation of Iranian Leishmania species at the species level, indicating conservation of these genes in the Leishmania parasite. Conclusion: The highest number of haplotypes belonged to L. tropica, indicating that the genetic diversity of this species is higher than that of L. major. It was further confirmed that the MLST is a suitable method to examine genetic variation of Leishmania parasites with respect to evolutionary and epidemiological studies.

Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda.

The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

Antibiotyping, RAPD- and ERIC-PCR fingerprinting of Klebsiella pneumoniae clinical isolates at a tertiary reference hospital in Denpasar, Bali, Indonesia.

Background and Objectives: Klebsiella pneumoniae is a healthcare-associated infections agent and could be an extended spectrum ß-lactamase (ESBL) producer. Understanding the transmission of this bacterium in a hospital setting needs accurate typing methods. An antibiogram is used to detect the resistance pattern of the isolates. Random Amplified Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR are rapid, technically simple, and easy-to-interpret DNA typing methods. This study aimed to evaluate the use of antibiotyping, RAPD-, and ERIC-PCR to investigate the heterogeneity of K. pneumoniae isolated from clinical specimens. Materials and Methods: The antibiograms of 46 K. pneumoniae clinical isolates were determined by Vitek® 2 Compact. All isolates underwent RAPD-PCR using AP4 primer and ERIC-PCR using ERIC-2 primer. The dendrogram was generated using the GelJ software and analyzed to determine its similarity. The analysis of antibiogram and the molecular typing diversity index was calculated using the formula of the Simpson's diversity index. Results: About 71.7% of the isolates were ESBL-producers, and more than 80% of isolates were susceptible to amikacin, ertapenem, and meropenem. The antibiotyping produced 32 diverse types with DI = 0.964. In addition, the RAPD-PCR produced 47 different types with DI = 1, while ERIC-PCR was 46 (DI=0.999). Conclusion: Antibiotyping, RAPD- and ERIC-PCR showed powerful discrimination power among the isolates, supported the diversity of K. pneumoniae isolates in current study. These combination could be promising tools for clonal relationship determination, including in tracking the transmission of the outbreak's agent in hospital setting.

Application of pulsed-field gel electrophoresis for molecular identification of pathogenic Leptospira species in Iran: a rapid and reliable method.

Background and Objectives: Leptospirosis is a zoonotic disease caused by pathogenic Leptospira serovars. The genus Leptospira cannot differentiated by conventional techniques. However, identity determination of pathogenic serovar is precious of public health problems and epidemiological studies. Pulsed-field gel electrophoresis facilitates rapid identification of Leptospires to the serovar levels. Materials and Methods: In this study, we employed PFGE to evaluate 28 Leptospira isolates, with animal, human and environmental origin, obtained from Razi Vaccine and Serum Research Institute of Karaj, Iran. PFGE patterns of 28 Leptospira serovars were generated using the Not I restriction enzyme in comparison with the lambda ladder. Results: Out of 28 serovars evaluated, we identified 22 different pulsed types, designated P1-P22. Out of 22 pulse groups, 3 were found to be a common type, but others were a single Type. Groups consisting of the common type were P3, P9, P14, and P16. The results showed that the discriminatory index of PFGE by Not I enzyme was 0.99, demonstrating heterogeneous differentiation among serovar members. Conclusion: The PFGE methodology used in this study showed excellent interlaboratory report usability, rapid, reliable, enabling standardization and data sharing between laboratories.

Molecular methods enhance the detection of pyoderma-related Streptococcus pyogenes and emm-type distribution in children.

BACKGROUND: Streptococcus pyogenes-related skin infections are increasingly implicated in the development of rheumatic heart disease (RHD) in lower-resourced settings, where they are often associated with scabies. The true prevalence of S. pyogenes-related pyoderma may be underestimated by bacterial culture. METHODS: A multiplex qPCR for S. pyogenes, Staphylococcus aureus and Sarcoptes scabiei was applied to 250 pyoderma swabs from a cross-sectional study of children <5 years in The Gambia. Direct PCR-based emm-typing was used to supplement previous whole genome sequencing (WGS) of cultured isolates. RESULTS: Pyoderma lesions with S. pyogenes increased from 51% (127/250) using culture to 80% (199/250) with qPCR. Compared to qPCR, the sensitivity of culture was 95.4% for S. pyogenes (95% CI 77.2-99.9) in samples with S. pyogenes alone (22/250, 9%), but 59.9% (95% CI 52.3-67.2) for samples with S. aureus co-infection (177/250, 71%). Direct PCR-based emm-typing was successful in 50% (46/92) of cases, identifying 27 emm-types, including six not identified by WGS (total 52 emm-types). CONCLUSIONS: Bacterial culture significantly underestimates the burden of S. pyogenes in pyoderma, particularly when co-infected with S. aureus. Molecular methods should be used to enhance the detection of S. pyogenes in surveillance studies and clinical trials of preventative measures in RHD-endemic settings.

Comparison of CRISPR typing and conventional molecular methods for distinguishing Laribacter hongkongensis isolates from fish, frogs and humans.

High-resolution and efficient typing for Laribacter hongkongensis (L. hongkongensis) is essential for epidemiological investigation of such emerging foodborne pathogens. Clustered regularly interspaced short palindromic repeats (CRISPR) typing is an innovative molecular method that shows great promise for L. hongkongensis typing. Here, we explored the CRISPR typing method by combining CRISPR1 and CRISPR2 loci to characterize a collection of 109 L. hongkongensis isolates from humans and animals and compared it to current molecular methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The results showed that all three methods have high discriminatory power (diversity index was 0.9902 for PFGE, 0.9663 for CRISPR and 0.9562 for MLST); strong congruence was observed between them (Rand index was 0.969 between CRISPR and PFGE, 0.953 between CRISPR and MLST, 0.958 between PFGE and MLST). CRISPR typing could well distinguish the isolates in the same STs or PFGE profiles, and the genetic information contained by the CRISPR array is useful for deep phylogenetic typing. We demonstrate that rapid CRISPR typing is a practical genetic fingerprinting tool with high resolution, comparable ease of use and lower cost, ability to track the source of various groups of L. hongkongensis strains and indication of genetic characteristics.