Restriction Enzyme Analysis Research Articles

First Genotyping of Mycobacterium avium Subspecies Paratuberculosis from Large Ruminant Population at Satna District in Madhya Pradesh in India to Bison Type

Background: Paratuberculosis or Johne’s disease in large ruminants is caused due to Mycobacterium avium subspecies paratuberculosis. The bacterial disease in large ruminants is characterized by chronic diarrhea, weakness and emaciation. There are no reports on Paratuberculosis in this area’s large ruminant population and its genotyping. Methods: A total of 76 samples (26 fecal and 50 milk) of large ruminants from Satna district in Madhya Pradesh were investigated (microscopical; for the presence of acid-fast organisms and molecular methods; for gene-specific amplification) for the presence of Mycobacterium avium subspecies paratuberculosis infection. Result: Microscopical examinations of fecal samples revealed 11.54% positivity and milk samples 14% positivity of Mycobacterium avium subspecies paratuberculosis infection. A single fecal and 2 milk samples were found positive in the IS900 polymerase chain reaction test (413 bp product). Specific amplification of 608 bp IS1311 product followed by restriction endonuclease analysis proved the genotype of Mycobacterium avium subspecies paratuberculosis as Bison type.

Epidemiology of Clostridioides difficile infection at one hospital 10 years after an outbreak of the epidemic C. difficile strain BI/027: changing strain prevalence, antimicrobial susceptibilities, and patient antibiotic exposures.

In contrast to the epidemiology 10 years earlier at our hospital when the epidemic restriction endonuclease analysis (REA) group strain BI accounted for 72% of Clostridioides difficile isolates recovered from first-episode C. difficile infection (CDI) cases, BI represented 19% of first-episode CDI isolates in 2013-2015. Two additional REA group strains accounted for 31% of isolates (Y, 16%; DH, 12%). High-level resistance to fluoroquinolones and azithromycin was more common among BI isolates than among DH, Y, and non-BI/DH/Y isolates. Multivariable analysis revealed that BI cases were 2.47 times more likely to be associated with fluoroquinolone exposure compared to non-BI cases (95% confidence interval [CI]: 1.12-5.46). In addition, the odds of developing a CDI after third- or fourth-generation cephalosporin exposure was 2.83 times for DH cases than for non-DH cases (95% CI: 1.06-7.54). Fluoroquinolone use in the hospital decreased from 2005 to 2015 from a peak of 113 to a low of 56 antimicrobial days/1,000 patient days. In contrast, cephalosporin use increased from 42 to 81 antimicrobial days/1,000 patient days. These changes correlated with a decrease in geometric mean MIC for ciprofloxacin (61.03 to 42.65 mg/L, P = 0.02) and an increase in geometric mean MIC for ceftriaxone (40.87 to 86.14 mg/L, P < 0.01) among BI isolates. The BI strain remained resistant to fluoroquinolones, but an overall decrease in fluoroquinolone use and increase in cephalosporin use were associated with a decrease in the prevalence of BI, an increased diversity of C. difficile strain types, and the emergence of strains DH and Y.

Identification of Nontuberculous Mycobacterium Species by Polymerase Chain Reaction - Restriction Enzyme Analysis (PCR-REA) of rpoB gene in Clinical Isolates.

Nontuberculous mycobacteria (NTM) infections are an emerging global health concern with increasing incidence. Conventional identification methods for NTM species in clinical settings are prone to errors. This study evaluates a newer method, polymerase chain reaction-restriction enzyme analysis (PCR-REA) of the rpoB gene, for NTM species identification. The study identified NTM species in clinical samples using conventional biochemical techniques and compared the results with PCR-REA of the rpoB gene. This cross-sectional study was conducted at a tertiary health-care center in North India over 18 months, analyzing both pulmonary and extrapulmonary samples. Two hundred and forty-seven NTM isolates were identified using phenotypic and biochemical methods. The same isolates were subjected to rpoB gene amplification by PCR followed by REA using Msp I and Hae III enzymes. Conventional methods identified 12 different NTM species (153 slow-growing and 94 rapid-growing), whereas PCR-REA identified 16 species (140 slow-growing, 107 rapid-growing). The Mycobacterium avium intracellulare complex was the most common species isolated. PCR-REA demonstrated higher resolution in species identification, particularly in differentiating within species complexes. PCR-REA of the rpoB gene proves to be a simple, rapid, and more discriminative tool for NTM species identification compared to conventional methods. This technique could significantly improve the diagnosis and management of emerging NTM infections in clinical settings.

Single Nucleotide Polymorphism and Restriction Enzyme Analysis of Chicken’s Prolactin Gene based on Genbank Sequences

Abstract The prolactin gene (PRL) has a role in controlling broodiness and egg production in chickens. This study aimed to identify Single Nucleotide Polymorphism (SNP) in the chicken PRL gene sequences and analyzed the possible restriction enzymes to conduct the PCR-RFLP as a preliminary study. Four sequences of the PRL gene were collected from GenBank and aligned. Primers were designed for the simulation of restriction enzyme analysis. Virtual PCR-RFLP gel electrophoresis was generated specifically for SNPs located in exon regions. As a result, there were 6 SNPs found in the exon regions. Restriction enzyme analysis revealed there were two SNPs (g.5806C/T and g.5856T/A) that cannot recognized by any restriction enzyme. The SNP g.5878C/T was only perceived by the TaqI enzyme. Three restriction enzymes could be used to recognize the SNP g.1621G/C (BsmAI, BsaI, and BcoDI) and g.5753G/C (SfaNI, HphI, and MslI). The BsrI, BmrI, BsMFI, and Hpy188I enzymes could be used to recognize SNP g.5781G/C. The SfaNI, HphI, and TaqI match the criteria for detection of the SNP g.5753 G/C and g.5878C/T using the PCR-RFLP method. In conclusion, the SfaNI, HphI, and TaqI enzymes can be used for conventional analysis using the PCR-RFLP method.

Estamaran date vinegar: chemical and microbial dynamics during fermentation.

Vinegar is a fermented food produced by alcoholic and then acetic acid microbial metabolism. Date palm fruit (Phoenix dactylifera L.) is a valuable source for the production of vinegar. Microbial identification has a major role in the improvement and bio-management of the fermentation process of vinegar. Estamaran and Kabkab two varieties of date palm fruit were selected to study the fermentation process. A culture-dependent approach was used to study bacterial dynamics. 16S rRNA gene was amplified by Polymerase Chain Reaction (PCR), also restriction enzyme analysis with HinfI and TaqI, and sequencing was done. Assessment of microbial flora of date palm fruit during fermentation showed that Fructobacillus tropaeoli, Bacillus sp., Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, and Weissella paramesenteroides existed in the first phase of fermentation. With fermentation progress, microbial diversity decreased so only one species remained. Komagataeibacter xylinus as an acid acetic producer was present in the third phase of fermentation. Based on chemical analysis, the concentration of reducing sugars decreased during fermentation. With decreasing pH, a simultaneous increase in acidity and total phenolic compounds occurred. The trend of changes during Estamaran fermentation was more severe and a vinegar with desirable properties was produced. Therefore, this date variety is recommended for the production of date vinegar.

Стан вивчення поліморфізму окремих генів у виникненні бронхіальної астми

The study of the polymorphism of individual genes is the basis of both preventive medicine and is used in the selection of adequate therapy. Polymorphism of individual candidate genes in bronchial asthma (BA) is studied taking into account many personal characteristics and the influence of environmental factors. Aim - to analyze the status of the study of the role of polymorphism of β2-adrenoceptors (ADRβ2) in the pathogenesis of bronchial asthma in children and to compare them with our own findings. Materials and methods. The results of research on polymorphism of individual genes in children with BA, which includes the PubMed database, were analyzed. An in-depth study of the anamnesis data (disease, life, hereditary), a complex clinical and laboratory examination of children (n=101) of school age with BA was carried out (the average age was: for boys 11.15±0.48 years; for girls - 10.63±0.7 years). Patients were diagnosed with BA in accordance with the GINA recommendations of the current revision. Determination of the Arg16Gly polymorphism of the ADRβ2 gene (rs 1042713) was performed by the polymerase chain reaction method followed by restriction enzyme analysis. The obtained data were processed in the Statsoft STATISTICA software package. Mean values were given as (M±m), Student's test was used for comparison. Results. The assessment of the frequency of polymorphism of the Arg16Gly gene in BA established that two variants prevailed in boys: Arg16Gly and Gly16Gly; at that time, the Gly16Gly genotype prevailed among girls, which, according to individual studies, is associated with good effectiveness of the applied therapy (nebulization of salbutamol). Mutations in the ADRβ2 gene in patients with BA were twice as frequent in the Arg16Gly genotype and three times as frequent in the Gln27Glu genotype, in contrast to healthy children. Such a polymorphism affects the functional activity of ADRβ2, the occurrence and course of BA. Conclusions. The Arg16Gly polymorphism of the ADRβ2 gene in children with BA largely determines the sensitivity of ADRβ2 to the applied exacerbation therapy and the effectiveness of basic treatment. In patients with the Gly16Gly genotype, most researchers note the good clinical effectiveness of both fast broncholytic drugs (β2 short-acting agonists) and basic anti-inflammatory drugs. The diagnosed homozygous Arg16Arg genotype of the ADRβ2 gene mostly accompanies the uncontrolled (or partially controlled) course of asthma in children. The revealed features of the Arg16Gly polymorphism of the ADRβ2 gene will help the doctor to draw up an adequate treatment plan, which is based on the genetically determined sensitivity of ADRβ2 receptors to medical preparations. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the author.

Genetic Variants of the Beta-Adrenergic Receptor Pathways as Both Risk and Protective Factors for Retinopathy of Prematurity

PurposeThere is strong evidence that genetic factors influence retinopathy of prematurity (ROP), a neovascular eye disease. It has been previously suggested that polymorphisms in the genes involved in beta-adrenergic receptor (ADRβ) pathways could protect against ROP. Antagonists for the ADRβ are actively tested in clinical trials for ROP treatment but not without controversy and safety concerns. This study was designed to assess if genetic variations in components of the ADRβ signaling pathways associate with risk of developing ROP. DesignAn observational case-control targeted genetic analysis. MethodsA study was carried out on premature participants with (n=30) or without (n=34) ROP and full-term controls (n=20) which were divided into a discovery cohort and a validation cohort. ROP was defined using International Classification of Retinopathy of Prematurity criteria (ICROP). Targeted sequencing of 20 genes in the ADRβ pathways was performed on the discovery cohort. Polymerase chain reaction (PCR)/restriction enzyme analysis for some of the discovered ROP-associated variants was performed for validation of the results using the validation cohort. ResultsThe discovery cohort revealed 543 bi-allelic variants within 20 genes of the ADRβ pathways. Ten single nucleotide variants (SNVs) in five genes including protein kinase A regulatory subunit 1α (PRKAR1A), rap guanine exchange factor 3 (RAPGEF3), adenylyl cyclase 4 (ADCY4), ADCY7, and ADCY9 were associated with ROP (p<0.05). The most significant SNV was found in PRKAR1A (p=0.001). Multiple variants located in the 3’-untranslated region (3’UTR) of RAPGEF3 were also associated with ROP (p<0.05). PCR/restriction enzyme analysis of the 3’UTR of RAPGEF3 methodologically validated these findings. ConclusionSNVs in PRKAR1A may represent protective factors while SNVs in RAPGEF3 may represent risk factors for ROP. PRKAR1α has previously been implicated in retinal vascular development while the RAPGEF3 product has a role in the maintenance of vascular barrier function, two processes important in ROP. Multicenter validation of these newly discovered risk factors could lead to valuable tools for predicting and preventing the development of severe ROP.

Comparative restriction enzyme analysis of methylation (CREAM) reveals methylome variability within a clonal in vitro cannabis population.

The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically uniform products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid multiplication of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to study a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

Use of genome-wide DNA methylation analysis to identify prognostic CpG site markers associated with longer survival time in dogs with multicentric high-grade B-cell lymphoma.

DNA methylation analysis might identify prognostic CpG sites in CHOP-treated dogs with multicentric high-grade B-cell lymphoma (MHGL) with heterogenous prognosis. To identify prognostic CpG sites of MHGL through genome-wide DNA methylation analysis with pyrosequencing validation. Test group: 24 dogs. Validation group: 100 dogs. All client-owned dogs were diagnosed with MHGL and treated with CHOP chemotherapy. Cohort study. DNA was extracted from lymph node samples obtained via FNA. Genome-wide DNA methylation analysis using Digital Restriction Enzyme Analysis of Methylation (DREAM) was performed on the test group to identify differentially methylated CpG sites (DMCs). Bisulfite pyrosequencing was used to measure methylation status of candidate DMCs in the validation group. Median survival times (MST) were analyzed using Kaplan-Meier (log-rank) product limit method. DREAM analyzed 101 576 CpG sites. Hierarchical clustering of 16 262 CpG sites in test group identified group with better prognosis (MST = 55-477 days vs 10-301 days, P = .007). Volcano plot identified 1371 differentially methylated CpG sites (DMCs). DMC near the genes of FAM213A (DMC-F) and PHLPP1 (DMC-P) were selected as candidates. Bisulfite-pyrosequencing performed on validation group showed group with methylation level of DMC-F < 40% had favorable prognosis (MST = 11-1072 days vs 8-1792 days, P = .01), whereas group with the methylation level combination of DMC-F < 40% plus DMC-P < 10% had excellent prognosis (MST = 18-1072 days vs 8-1792 days, P = .009). Methylation status of prognostic CpG sites delineate canine MGHL cases with longer MST, providing owners with information on expectations of potential improved treatment outcomes.

The serological and genetic diversity of the Leptospira interrogans Icterohaemorrhagiae serogroup circulating in the UK.

Strains of Leptospira interrogans belonging to two very closely related serovars, Icterohaemorrhagiae and Copenhageni, have been associated with disease in mammalian species and are the most frequently reported agents of human leptospirosis. They are considered the most pathogenic serovars and represent more than half of the leptospires encountered in severe human infections. Nineteen such isolates from the United Kingdom - human, domestic and wildlife species - were typed using three monoclonal antibodies (F12 C3, F70 C14 and F70 C24) in an attempt to elucidate their epidemiology. They were further examined by restriction endonuclease analysis (REA), multiple-locus variable-number tandem repeat analysis (MLVA) and lic12008 gene sequence analysis. Monoclonal antibody F12 C3, which is highly specific for Icterohaemorrhagiae and Copenhageni, confirmed that all the strains belonged to these two serovars. Sixteen strains were identified as Copenhageni and three as Icterohaemorrhagiae serovar. Only one restriction pattern type was identified, thus confirming that REA is not able to discriminate between the Icterohaemorrhagiae and Copenhageni serovars. Variable-number tandem-repeat analysis found three loci with differences in the repeat number, indicating genetic diversity between British isolates. Sequences of the lic12008 gene showed that all isolates identified as the Icterohaemorrhagiae serotype have a single base insertion, in contrast to the same sequences of the Copenhageni serotype. Copenhageni is the predominant serovar in the Icterohaemorrhagiae serogroup isolated in British Isles. There is a genetic diversity of MLVA patterns of the isolates but no genetic tool used in the study was able to determine serovars.

Error-Corrected Next-Generation Sequencing Provides a Comprehensive Overview of the Subclonal Mutation Landscape and Its Prognostic Implications in Juvenile Myelomonocytic Leukemia

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myelodysplastic/myeloproliferative neoplasm characterized by mutations in genes activating the RAS pathway ( PTPN11, NF1, NRAS, KRAS, and CBL). Approximately 20% of patients with JMML have secondary mutations in SETBP1 and/or JAK3, and recent droplet digital PCR (ddPCR) analyses revealed that mutations in these two genes, including very rare mutations (variant allele frequency [VAF] &amp;lt; 0.05) that are difficult to detect by conventional next-generation sequencing (NGS), are associated with poor prognoses. Meanwhile, the landscape of low VAF subclonal mutations of other genes in patients with JMML has not been adequately evaluated. Therefore, we utilized error-corrected NGS, which can reduce the systematic sequencing error rate of conventional NGS by incorporating molecular barcodes (MBCs). Methods: We performed error-corrected NGS in 93 patients with JMML and 11 patients with JMML-like myeloproliferative disorder with Noonan syndrome (NS/MPD) using a small panel associated with myeloid malignancies. Samples for error-corrected NGS were prepared using a SureSelect XT HS2 DNA Library Preparation Kit (Agilent Technologies), including unique dual index and duplex MBCs. A unique MBC is incorporated into each DNA fragment, and the incorporated MBC can differentiate true and false duplications, allowing for highly accurate sequencing that can detect mutations with VAF &amp;gt; 0.001. We used the HiSeq X platform (Illumina) and SureCall v.4.2 application (Agilent Technologies) for analysis. In addition, DNA methylation analysis was performed on the same cohort using the Infinium HumanMethylation 450K BeadChip and/or Digital Restriction Enzyme Analysis of Methylation as previously described (Murakami et al. 2018;131:1576-1586, Kitazawa et al. Blood Adv. 2021;5:5507-5518). This study was approved by the Ethics Committee of the Nagoya University Graduate School of Medicine. Results: In the cohort of 104 patients with JMML or NS/MPD, 160 mutations were identified, of which 30 (19%) had VAF &amp;lt; 0.05 (eight in JAK3, seven in NF1, five in ASXL1, three in NRAS, two each in PTPN11 and SOS1, and one each in SETBP1, SH2B3, and ZRSR2). Subclonal mutations in SETBP1 p.D868N and JAK3 p.R657Q identified by error-corrected NGS were confirmed using ddPCR, and consistent results were obtained for both assays (R 2 = 0.9977 and 0.9919 respectively). Of 93 patients with JMML, 30 (32%) had subclonal or clonal secondary mutations, whereas no such mutations were detected in patients with NS/MPD ( P = 0.031). Of 104 patients, 23 (22%) had secondary mutations with VAF ≥ 0.05 (Major group), 7 (7%) only had secondary mutations with VAF &amp;lt; 0.05 (Minor group), and the remaining 74 (71%) had no secondary mutations (Negative group). We assessed the associations of subclonal mutations detected by error-corrected NGS with the DNA methylation profile, the most important prognostic factor in JMML. Whereas 22 of 23 (96%) and 5 of 7 (71%) patients in the Major and Minor groups, respectively, had high methylation (HM) profiles, only 20 of 74 (27%) patients in the Negative group featured HM profiles ( P &amp;lt; 0.001). The 4-year overall survival (OS) rates were significantly lower in the Major (52.2% [95% confidence interval {CI} = 30.5%-70.0%], P = 0.01) and Minor groups (42.9% [95% CI = 9.8%-73.4%], P = 0.03) than in the Negative group (76.3% [95% CI = 64.6%-84.5%]). In a subgroup analysis of 30 patients with secondary mutations, patients with secondary mutations in RAS pathway genes (n = 18) had significantly lower 4-year OS rates than those with secondary mutations in other genes (n = 12; 32.4% [95% CI = 12.7%-54.0%] vs. 75.0% [95% CI: 40.8%-91.2%], P = 0.047). Conclusions: We successfully performed error-corrected NGS to assess comprehensive subclonal secondary mutational profiles, including very low VAF variants. The presence of subclonal mutations, particularly in RAS pathway genes, was associated with poor OS. These findings provide important information for appropriate risk stratification, which will contribute to the implementation of precision medicine for patients with JMML.

Evaluation of the polymorphism of the GDF9 and BMP15 genes and their relationship with the reproductive functions of sheep of different breeds

Aim. Polymorphism is the result of evolutionary processes and is inherited, associated with biodiversity and modified by natural selection and functions to preserve various forms of population. It is economically advantageous to find genes that can be used in breeding in such a way as to increase the fertility of animals in different natural geographical zones, since the adaptive abilities of organisms are complicated. In this regard, the purpose of this study was to study the genetic potential of sheep of different breeds bred in various ecological and geographical regions of the North Caucasus with pasture-pasture and pasture-stall conditions of sheep breeding.Material and Methods. Genotyping was undertaken of sheep of different breeds contained in various natural and climatic zones of the Republic of Dagestan and the most arid region of Stavropol Territory in the zone of risky agriculture where the climate is sharply continental. Genotyping in the loci of the genes of differential growth factor (GDF9) and bone morphogenetic protein (BMP15) was carried out by PCR-PDRF (polymorphism of the lengths of restriction fragments) by cutting DNA using endonuclease restriction and further analysis of the size of the resulting PCR fragments. The genetic structure of genes has been studied by methods of genetic and statistical analysis of the data obtained.Conclusion. The genetic structure of sheep populations of different breeds bred in the foothills of the Republic of Dagestan and the arid region of Stavropol Territory was studied for the first time. Information obtained on the role of the degree of genetic variability of sheep of different breeds provides data which can contribute to the ecological well-being of herds and the status characterising the signs of multiple birth rates is also determined. This will further improve and significantly accelerate breeding work with livestock, since an important task of further increasing the efficiency of the industry is the reproduction of the herd together with a simultaneous increase in the productivity of animals.

Plasmid and Cell Envelope Characterization of &lt;i&gt;Vibrio alginolyticus&lt;/i&gt; Isolated from Infected Tiger Prawn,&lt;i&gt;Penaeus monodon&lt;/i&gt;

It has been reported that indiscriminate use of antibacterial compounds in aquaculture has resulted in an increase in populations of antibiotic resistant strains of bacteria. In the present study, Vibrio alginolyticus isolated from infected Penaeus monodon has been found resistant to Furoxone (30 g), Ampicillin (10 g), Cloxacillin (1 g), Bacitracin (10 units), Penicillin (10 units) and Oxytetracycline (30 pg) by antibiotic sensitivity test, suggesting the restricted prophylactic use of antibiotics in shrimp aquaculture industry. As an approach towards differentiation and identification of bacterial strain, the plasmid characterization of V. alginolyticus by restriction enzyme analysis was carried out which revealed the presence of 6.3 kb and 0.8 kb DNA fragments by Hind lil and a 7. 9 kb DNA fragment by EcoRI enzymes. The cell envelope characterization of V. alginolyticus by SOS-PAGE showed the presence of 12 protein bands in the range of 29 to 112.20 kD. The preliminary information on the cell envelope characterization may help in studies relating towards development of immunostimulant against vibriosis.

Preliminary study on local Indonesian sheep MC4R gene as a prospective marker for sustainable high production trait

A prospective genetic marker to archive for sustainable growth traits of local Indonesian sheep is the Melanocortin-4 Receptor (MC4R) gene, which arrange feeding behavior. This study were examined the MC4R gene of Indonesian sheep for polymorphism and restriction enzyme for PCR-RFLP within the partial coding sequence (CDS) and 3’UTR regions. A 642 bp of PCR product was amplified from sixteen blood samples (Sakub: 2, Wonosobo: 3, Sapudi: 3, Garut:3, and Garut-Dorper cross: 2). Sequence alignments between the samples and GenBank (NC_056076) discovered five SNPs (CDS: SNPs 1039A>G and 1068G>C, 3’UTR region: SNPs 1403G>A, 1511C>A and 1526G>C). The SNP 1039A>G was categorized as a missense mutation (Met to Val). However, SNP 1068G>C was a silent mutation (Ala to Ala). The restriction enzyme analysis showed that SNPs 1039A>G and 1403G>A were recognized by BssSI and SfaNI. SNPs 1068G>C, 1511C>A and 1526G>C were not recognized by the enzyme. In conclusion, the MC4R gene of Indonesian has five SNPs discovered, among other two SNPs in CDS and three SNPs in 3’UTR region. BssSI and SfaNI enzymes are eligible for further genotyping of sheep samples using the PCR-RFLP method.

Characteristics and outcomes of Clostridioides difficile infection after a change in the diagnostic testing algorithm.

Polymerase chain reaction (PCR) testing for the detection of C. difficile is a highly sensitive test. Some clinical laboratories have included a 2-step testing algorithm utilizing PCR plus toxin enzyme immunoassays (EIAs) to increase specificity. To determine the risk factors and outcomes of C. difficile PCR-positive/toxin-positive encounters compared to PCR-positive/toxin-negative encounters. Retrospective study. A Veterans' Affairs hospital. A retrospective case-control study of patient encounters with a positive C. difficile test by PCR and either a toxin EIA-positive assay (ie, cases) or toxin EIA-negative assay (ie, controls). Clinically relevant exposures and risk factors were determined to assess CDI recurrence at 30 days. Available encounter stool specimens were cultured for C. difficile and were subjected to restriction endonuclease analysis (REA) strain typing. Among 130 C. difficile PCR-positive patient encounters, 80 (61.5%) were toxin EIA negative and 50 (38.5%) were toxin EIA positive. Encounters that were toxin positive were more frequently treated (96.0%) compared to toxin-negative encounters (71.3%; P < .01). A multivariable logistic regression model revealed that toxin-negative encounters were less likely to suffer a recurrent CDI episode within 30 days (odds ratio [OR], 0.20, 95% confidence interval [CI], 0.05-0.83). Additionally, a higher C. difficile PCR cycle threshold predicted a lower risk of CDI recurrence at 30 days. (OR, 0.82; 95% CI, 0.68-0.98). During the study period, the REA group Y strain accounted for most toxin-negative encounters (32.5%; P = .05), whereas REA group BI strain accounted for most toxin-positive encounters (24.3%; P = .02). A testing strategy of PCR plus toxin EIA helped predict recurrent CDI.

Cord Blood Hematological Parameters of Fetuses Detected Different Thalassemia Genotypes in the Second Trimester of Pregnancy

Hemoglobinopathies are the most common inherited diseases in humans resulting from impaired globin chain synthesis of hemoglobin. The progression of thalassemia rates is prevented with prenatal screening methods. To evaluate the hematological parameters of α- and β-thalassemia and normal fetuses aged 17-25 weeks of gestation. A cross-sectional study. Pregnant women who underwent cordocentesis in the second trimester because of the risk of having a baby with thalassemia were included in the study. Hematological indices and molecular DNA methods were analyzed from the cord blood samples of 129 women who were 17-25 weeks into pregnancy. The HPLC method was used for Hb fraction analysis. Amplification refractory mutation system, restriction enzyme analysis, multiplex polymerase chain reaction, and sequencing methods were used for the molecular analysis. Maternal contamination was eliminated by the short tandem repeat method. In total, 112 of the fetuses carry α- and β-thalassemia heterozygous or homozygous (α: 37, β: 58, mixed: 17) and 17 fetuses had a normal genotype for thalassemia. Significant differences in adult hemoglobin (HbA), fetal hemoglobin (HbF), Hb Barts, MCV, MCH, and RDW were detected in three groups compared with the normal group (p < 0.001, except for RBC, Hb, HCT, and MCHC). Differences in HbF, Hb Barts, MCV, MCH, and RDW were observed in the α-thalassemia groups compared with the normal group (p < 0.001). Among the five β-thalassemia subgroups, only HbA and RDW were different from the normal group (p < 0.001). This study could be a good reference for future studies and prenatal diagnostic applications in emphasizing the importance of changes in the blood parameters of fetuses before molecular genotyping. These hematological data give valuable information to clinicians about the fetus to enlighten families in making appropriate decisions during prenatal diagnosis.

Effect of the COVID-19 Pandemic on Rates and Epidemiology of Clostridioides difficile Infection in One VA Hospital.

The COVID-19 pandemic was associated with increases in some healthcare-associated infections. We investigated the impact of the pandemic on the rates and molecular epidemiology of Clostridioides difficile infection (CDI) within one VA hospital. We anticipated that the potential widespread use of antibiotics for pneumonia during the pandemic might increase CDI rates given that antibiotics are a major risk for CDI. Hospital data on patients with CDI and recurrent CDI (rCDI) were reviewed both prior to the COVID-19 pandemic (2015 to 2019) and during the pandemic (2020-2021). Restriction endonuclease analysis (REA) strain typing was performed on CD isolates recovered from stool samples collected from October 2019 to March 2022. CDI case numbers declined by 43.2% in 2020 to 2021 compared to the annual mean over the previous 5 years. The stool test positivity rate was also lower during the COVID-19 pandemic (14.3% vs. 17.2%; p = 0.013). Inpatient hospitalization rates declined, and rates of CDI among inpatients were reduced by 34.2% from 2020 to 2021. The mean monthly cases of rCDI also declined significantly after 2020 [3.38 (95% CI: 2.89-3.87) vs. 1.92 (95% CI: 1.27-2.56); p = <0.01]. Prior to the pandemic, REA group Y was the most prevalent CD strain among the major REA groups (27.3%). During the first wave of the pandemic, from 8 March 2020, to 30 June 2020, there was an increase in the relative incidence of REA group BI (26.7% vs. 9.1%); After adjusting for CDI risk factors, a multivariable logistic regression model revealed that the odds of developing an REA group BI CDI increased during the first pandemic wave (OR 6.41, 95% CI: 1.03-39.91) compared to the pre-pandemic period. In conclusion, the incidence of CDI and rCDI decreased significantly during the COVID-19 pandemic. In contrast, REA BI (Ribotype 027), a virulent, previously epidemic CD strain frequently associated with hospital transmission and outbreaks, reappeared as a prevalent strain during the first wave of the pandemic, but subsequently disappeared, and overall CDI rates declined.

Antimicrobial susceptibility of Clostridioides difficile to omadacycline and comparator antimicrobials.

Omadacycline is a novel aminomethylcycline tetracycline antimicrobial that was approved for the treatment of community-associated bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI) in 2018. Omadacycline has demonstrated a high degree of in vitro activity towards Clostridioides difficile and previous data have hypothesized that use of omadacycline for CABP or ABSSSI may decrease the risk of C. difficile infections. To compare the in vitro antimicrobial activity of omadacycline versus commonly used antimicrobials for the approved indications of use. We compared the antimicrobial activity of eight antimicrobials approved for CABP and ABSSSI against omadacycline by agar dilution on 200 clinically relevant contemporary C. difficile isolates representing local and national prevalent strain types. The in vitro omadacycline geometric mean MIC was 0.07 mg/L. Ceftriaxone resistance was noted in >50% of all isolates tested. The epidemic strain group, identified as restriction endonuclease analysis (REA) group BI, was commonly resistant to azithromycin (92%), moxifloxacin (86%) and clindamycin (78%). REA group DH strains had an elevated trimethoprim/sulfamethoxazole geometric mean MIC of 17.30 mg/L compared with the geometric mean MIC of 8.14 mg/L noted in all other isolates. In the REA group BK isolates that had a doxycycline MIC of ≥2 mg/L, the omadacycline MIC was <0.5 mg/L. Among 200 contemporary C. difficile isolates, there were no notable elevations in the in vitro omadacycline MIC, indicating a high level of activity towards C. difficile in comparison with commonly used antimicrobials for CABP and ABSSSI.

Penicillin-binding protein genotyping of penicillin-nonsusceptible Streptococcus pneumoniae isolates from the nasopharynx of healthy preschool children

Abstract Streptococcus pneumoniae is one of the most frequent bacterial identified causes of community-acquired pneumonia, otitis media and meningitis. It is, as well, a common cause of bacteraemia’s significant morbidity and mortality. Beta-lactam antibiotics (BLAs) are the first line of empirical treatment for pneumococcal infections. The targets of BLAs are penicillin-binding proteins (PBPs), the modifications of which are one of the reasons why pneumococci are non-susceptible to BLAs. In our work, a total of 39 Streptococcus pneumoniae isolates were obtained from 176 healthy children, both vaccinated and non-vaccinated. The isolates were tested for antimicrobial susceptibility, and their penicillin-binding proteins (PBPs) were typed by the restriction fragment length of the polymorphism analysis of their pbp genes. The most frequent serotypes among the penicillin non-susceptible Streptococcus pneumoniae (PNSSP) isolates were 23B, 35B and 19F. Restriction enzyme analyses of pbp1a, pbp2b, and pbp2x genes revealed 5, 3 and 3 different patterns, respectively, and a total of 4 different PBPs profiles of PNSSP isolates belonging to serotypes not included in pneumococcal conjugate vaccines were demonstrated. We conclude that the level of resistance should be monitored constantly to ascertain the effect of current pneumococcal conjugate vaccines, as well as to recognize new circumstances developing in Poland, as well as the possibility of multiple, independent imports of resistant strains from abroad.

Biomolecular Analysis of Canine Distemper Virus Strains in Two Domestic Ferrets (Mustela putorius furo)

Canine distemper is a contagious and severe systemic viral disease that affects domestic and wild carnivores worldwide. In this study, two adult female ferrets (Mustela putorius furo) were evaluated for cutaneous lesions. Scab, fur, and swab samples from the external auditory canal, cutaneous lesions, and scrapings were analyzed. Canine distemper virus (CDV)-positive samples underwent RT-PCR/RFLP with the restriction enzyme PsiI, and the hemagglutinin gene sequence was obtained. According to the restriction enzyme and sequence analyses, the viral strains were typed as CDV field strains that are included within the Europe lineage and distinct from those including vaccinal CDV strains. The sequence analysis showed the highest nucleotide identity rates in older Europe lineage CDV strains collected from dogs and a fox in Europe. This study is the first to report on CDV infection in ferrets in southern Italy and contributes to the current knowledge about natural CDV infection in this species. In conclusion, vaccination remains crucial for preventing the disease and counteracting cross-species infection. Molecular biology techniques can enable the monitoring of susceptible wild animals by ensuring the active surveillance of CDV spread.