Polymerase Chain Reaction Products Research Articles

High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens.Graphical

Utility of Cerebrospinal Fluid Unstimulated Interferon-Gamma (IRISA-TB) as a Same-Day Test for Tuberculous Meningitis in a Tuberculosis-Endemic, Resource-Poor Setting.

Tuberculous meningitis (TBM) mortality is high and current diagnostics perform suboptimally. We evaluated the diagnostic performance of a DNA-based assay (GeneXpert Ultra) against a new same-day immunodiagnostic assay that detects unstimulated interferon-gamma (IRISA-TB). In a stage 1 evaluation, IRISA-TB was evaluated in biobanked samples from Zambia (n = 82; tuberculosis [TB] and non-TBM), and specificity in a South African biobank (n = 291; non-TBM only). Given encouraging results, a stage 2 evaluation was performed in suspected TBM patients from Zimbabwe and Malawi (n = 668). Patients were classified as having definite, probable or possible TBM, or non-TBM based on their microbiological results, cerebrospinal fluid (CSF) chemistry, and whether they received treatment. In the stage 1 evaluation, sensitivity and specificity of IRISA-TB were 75% and 87% in the Zambian samples, and specificity was 100% in the South African samples. In the stage 2 validation, IRISA-TB sensitivity (95% confidence interval [CI]) was significantly higher than Xpert Ultra (76.2% [55.0%-89.4%] vs 25% [8.9%-53.3%]; P = .0048) when trace readouts were considered negative. Specificity (95% CI) was similar for both assays (91.4% [88.8%-93.4%] vs 86.9% [83.4%-89.8%]). When the Xpert Ultra polymerase chain reaction product was verified by sequencing, the positive predictive value of trace readouts in CSF was 27.8%. Sensitivity of IRISA-TB was higher in human immunodeficiency virus (HIV)-infected versus uninfected participants (85.8% vs 66.7%). As a same-day rule-in test, IRISA-TB had significantly better sensitivity than Xpert Ultra in a TB/HIV-endemic setting. An immunodiagnostic approach to TBM is promising, and further studies are warranted.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding

ObjectiveLeptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. MethodsForty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. ResultsWe revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. ConclusionWe repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

Isolation, identification and histopathological observation of Francisella noatunensis subsp. orientalis from Nile tilapia (Oreochromis niloticus)

In April 2023, a substantial mortality event struck the primary Nile tilapia (Oreochromis niloticus) breeding areas in Maoming City, Guangdong Province, resulting in considerable economic losses. To unravel the etiology of this disease outbreak, thorough farm field necropsies were conducted, encompassing detailed descriptions of gross clinical signs and diagnostic sample collection. Clinically, affected tilapia primarily manifested multifocal superficial skin ulcers, mild focal congestion, and hemorrhage in the jaws, eyes' irises, and other integumentary regions. Tissue specimens from diseased fish were screened for Francisella spp., using a polymerase chain reaction (PCR) assay. The PCR product targeting polymerase gene showed 99.64 % nucleotide sequence identity to the published sequence of Francisella noatunensis subsp. orientalis (F. noatunensis subsp. orientalis). Histopathological examination of selected tissues revealed acute to subacute, severe necrotizing bacterial splenitis accompanied by diffuse vasculitis, marked necrotizing nephritis, mild necrotizing hepatitis, and prominent proliferative vasculitis. Transmission electron microscopy (TEM) further demonstrated that F. noatunensis subsp. orientalis was predominantly found in macrophage cytoplasm, appearing as round, spheroidal bacteria. Koch's postulates were successfully confirmed through challenge testing. Moreover, F. noatunensis subsp. orientalis exhibited high sensitivity to Gentamicin, Vancomycin, Minocycline, Imipenem, and Ceftazidime. In conclusion, F. noatunensis subsp. orientalis has emerged as a significant pathogen affecting Nile tilapia in China, characterized by systemic multi-organ necrotizing vasculitis with the spleen and head kidney being the primary targets.

Diagnosis of avian salmonellosis using PCR kits of different manufacturers

Relevance. The problem in timely detection of this infection is that poultry contaminated with salmonellae may not show clinical signs of the disease. Polymerase chain reaction in the diagnosis of avian salmonellosis is designed for direct detection and identification of pathogenic agents, especially in cases where their detection is difficult by other diagnostic methods. Advantages of PCR tests allow to successfully use this method in veterinary laboratory practice. The aim of this work is to conduct comparative tests of diagnostic PCR kits of different manufacturers in the diagnosis of avian salmonellosis.Methods. To conduct comparative studies of pathological material for the presence of the DNA of Salmonella spp., Salmonella enteritidis and Salmonella typhimurium by molecular biological method, three commercial kits of domestic and imported production for isolation and detection of Salmonella DNA by PCR were used. Determination of the possibility of using membrane glass fiber materials, in the form of cards for taking dry samples of pathogenic material, for subsequent PCR was carried out in comparative tests with parallel studies of biomaterial delivered to the laboratory in frozen form.Results. Comparative studies of pathological material for the presence of Salmonella spp. DNA using three commercial kits for the isolation and detection of Salmonella DNA by polymerase chain reaction, imported and domestic production were carried out. It was found that when comparing the results obtained using kits of different manufacturers for the presence of Salmonella spp. DNA, comparable results were observed. The study was performed within the framework of the state assignment No. FGUG-2022-0009 of (FSC VIEV).

Genetic diversity, virulence profiles, and antimicrobial resistance of Salmonella enterica serovar Typhi isolated from typhoid fever patients in Baghdad, Iraq

Typhoid fever is an important health issue in developing countries, and the pathogenicity of Salmonella enterica serovar Typhi (S. ser. Typhi) depends on the presence of different virulence factors. Therefore, this study aimed to investigate the spread of virulence genes among S. Typhi isolates from patients with typhoid fever in Baghdad, Iraq. Sixty S. Typhi isolates were collected from several hospitals in Baghdad and identified using VITEK-II and confirmed by polymerase chain reaction (PCR) to detect the 16S rRNA gene. After testing their susceptibility to different antimicrobials (via the disk diffusion method), we found the highest resistance rates (100 %) were to ampicillin, piperacillin, cefotaxime, and ceftriaxone. The highest sensitivity rates (100 %) were to ertapenem, imipenem, meropenem, and sulfamethoxazole/trimethoprim. The presence of genes encoding for virulence in S. Typhi isolates was tested by conventional PCR. The results showed that out of 60 isolates, 59 (98.3 %), 59 (98.3 %), 58 (96.7 %), and 60 (100 %) were positive for viaB, staA, cdtB, and orfL genes, respectively. The sequencing of PCR products (viaB, staA, cdtB, and orfL genes) was carried out at the Macrogen Company (Seoul, Korea). The sequences were compared with nucleotide sequences in the BLAST GenBank database, and data obtained from the sequencing of these virulence genes were submitted to GenBank under different accession numbers. A phylogenetic analysis of the 16S rRNA gene sequence found a high similarity between local sequences and the closely related sequences of genes in GenBank. The presence of the viaB, staA, cdtB, and orfL virulence genes in nearly all of the isolates under examination suggests that they play an important role in the pathogenicity of local isolates.

Evidence of “Silent” Hepatitis B Virus Infection in Psoriasis, Vitiligo, and Pityriasis Rosea Cases: A Pilot Study

Abstract Background: Psoriasis (PS), vitiligo (VT), and Pityriasis rosea (PR) are chronic skin diseases often occurring as a consequence of exaggerated immune responses. These skin manifestations can be triggered as a result of the molecular mimicry between viral protein (s) and host protein (s), which could generate auto-antibodies. In addition, it can be hypothesised that skin diseases are manifestations of the reduced immunity that is observed in chronic hepatitis B virus (HBV)-infected individuals. Aims and Objective: To investigate the presence of HBV in PS, VT, and PR cases and Human Herpes Virus (HHV) 6 and 7 in PR cases. Materials and Methods: DNA extracted from healthy controls (n = 20), PS (n = 10), VT (n = 11), and PR (n = 12) were subjected to HBV-S gene-specific polymerase chain reactions (PCRs) and HHV 6-UL57 and HHV7-UL10 gene-specific PCRs. PCR products of positive samples (HBV and HHV 6 and 7 DNA) of expected length were bi-directionally sequenced using overlapping primers. Sequence identification was performed by NCBI BLAST and analysed by multiple sequence alignment. HBV DNA copy number was determined through quantitative real-time PCR. The blood samples were also tested for HBV serological markers and Interferon gamma (IFN-γ) by enzyme immunoassays. Results: The PCR data and Immunoassay study revealed that seven out of 12 PR, six out of 10 PS, and six out of 11 VT cases had signs of HBV infection. HHV 6 DNA was detected in four, whereas HHV 7 DNA was found in two of the 12 PR blood samples. PR6 presented the evidence of both HHV 6 and 7 co-infections. Conclusion: Observing the correlation of HBV with skin diseases, albeit at the pilot level, a larger study is warranted to identify HBV infection in skin disease patients. The evidence of HHV 6 and HHV 7 DNA in PR cases supports the HHV infection linkage with PR.

Pharmacogenetic Approach to Tramadol Use in the Arab Population.

Tramdol is one of most popular opioids used for postoperative analgesia worldwide. Among Arabic countries, there are reports that its dosage is not appropriate due to cultural background. To provide theoretical background of the proper usage of tramadol, this study analyzed the association between several genetic polymorphisms (CYP2D6/OPRM1) and the effect of tramadol. A total of 39 patients who took tramadol for postoperative analgesia were recruited, samples were obtained, and their DNA was extracted for polymerase chain reaction products analysis followed by allelic variations of CYP2D6 and OPRM A118G determination. Numerical pain scales were measured before and 1 h after taking tramadol. The effect of tramadol was defined by the difference between these scales. We concluded that CYP2D6 and OPRM1 A118G single nucleotide polymorphisms may serve as crucial determinants in predicting tramadol efficacy and susceptibility to post-surgical pain. Further validation of personalized prescription practices based on these genetic polymorphisms could provide valuable insights for the development of clinical guidelines tailored to post-surgical tramadol use in the Arabic population.

CagA 3' region polymorphism of Helicobacter pylori and its association with chronic gastritis in the Chinese population.

Introduction. Cytotoxin-associated gene A (CagA) from Helicobacter pylori is highly related to chronic gastritis. Tyrosine phosphorylation of Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs from CagA determines the pathogenicity of H. pylori.Gap statement. The precise amino acid variations surrounding the EPIYA motifs and their correlation with clinical outcomes have been poorly explored.Aim. The purpose of this study was to examine the CagA 3' region polymorphism of H. pylori and its association with chronic gastritis in the Chinese population.Method. A total of 86 cagA-positive H. pylori strains were isolated from patients with chronic gastritis in two different hospitals in Beijing, PR China. Genomic DNA was extracted commercial kits, and the cagA 3' variable region of H. pylori was amplified by polymerase chain reaction (PCR). The PCR products were sequenced and analysed using the CLC Sequence Viewer, BioEdit, and WebLogo 3.Results. Two hundred and fifty-nine EPIYA motifs were identified from cagA-positive H. pylori strains. Notably, EPIYA-B exhibited a higher frequency of variation in comparison to EPIYA-A, EPIYA-C, and EPIYA-D. The prevalent sequences for East-Asian-type CagA were QVNK and TIDF, while KVNK and TIDD were most commonly observed for Western-type CagA. The CRPIA motifs of East-Asian-type CagA and Western-type CagA varied at positions 4, 6, 7, 8, and 10. CagA-ABD (73.2%) was the most prevalent type, followed by CagA-ABC (18.6%) and CagA-AB (3.4%). The ratio of CagA-ABD was observed to be higher in cases of chronic non-atrophic gastritis with erosive (NAGE) or chronic atrophic gastritis (AG) compared to chronic non-atrophic gastritis (NAG), and the difference was found to be statistically significant (χ2=59.000/64.000, P<0.001).Conclusions. The EPIYA segments of Western-type CagA and East-Asian-type CagA differ significantly and the presence of CagA-ABD may be associated with severe chronic gastritis from this study.

Long-range and real-time PCR identification of a large SERPINC1 deletion in a patient with antithrombin deficiency.

Congenital antithrombin (AT) or serpin C1 deficiency, caused by a SERPINC1 abnormality, is a high-risk factor for venous thrombosis. SERPINC1 is prone to genetic rearrangement, because it contains numerous Alu elements. In this study, a Japanese patient who developed deep vein thrombosis during pregnancy and exhibited low AT activity underwent SERPINC1 gene analysis using routine methods: long-range polymerase chain reaction (PCR) and real-time PCR. Sequencing using long-range PCR products revealed no pathological variants in SERPINC1 exons or exon-intron junctions, and all the identified variants were homozygous, suggesting a deletion in one SERPINC1 allele. Copy number quantification for each SERPINC1 exon using real-time PCR revealed half the number of exon 1 and 2 copies compared with controls. Moreover, a deletion region was deduced by quantifying the 5'-upstream region copy number of SERPINC1 for each constant region. Direct long-range PCR sequencing with primers for the 5'-end of each presumed deletion region revealed a large Alu-mediated deletion (∼13kb) involving SERPINC1 exons 1 and 2. Thus, a large deletion was identified in SERPINC1 using conventional PCR methods.

Novel Gammaherpesvirus Infections in Narrow-Ridged Finless Porpoise (Neophocaena asiaeorientalis) and False Killer Whales (Pseudorca crassidens) in the Republic of Korea.

A female narrow-ridged finless porpoise (Neophocaena asiaeorientalis) stranded on a beach on Jeju Island showed epithelial proliferative skin lesions on its body. Two false killer whales (Pseudorca crassidens), caught using nets near Gangneung and Samcheok, respectively, had multiple plaques on their penile epidermis. Histological examination of the epidermis revealed that all the lesions had common features, including accentuated rete pegs, ballooning changes, and eosinophilic intranuclear inclusion (INI) bodies. Based on the histopathological results, herpesvirus infection was suspected, and thus further analysis was conducted using herpesvirus-specific primers. Based on nested polymerase chain reaction (PCR) tests using the herpesvirus-detectable primers, the PCR products demonstrated two fragments: a 222-base-pair (bp) sequence of the DNA polymerase gene, SNUABM_CeHV01, showing 96.4% identity with a bottlenose dolphin herpesvirus from the Jeju narrow-ridged finless porpoise; and a 222 bp sequence of the DNA polymerase gene, SNUABM_CeHV02, showing 95.95% identity with the same bottlenose dolphin herpesvirus from the Gangneung and Samcheok false killer whales. The significance of this study lies in its ability to demonstrate the existence of novel cetacean herpesviruses in South Korean seawater, representing an important step forward in studying potentially harmful pathogens that affect endangered whale and dolphin populations.

Mutation Analysis of Exon 1 in the Hemoglobin Subunit Beta (HBB) Gene in Beta-Thalassemia.

Introduction Thalassemia is a widely prevalent monogenic hematological disorder found worldwide. It exists in two forms: alpha- and beta-thalassemia. Alterations in the hemoglobin subunit beta (HBB) gene cause beta-thalassemia, with missense and point mutations affecting beta-globin synthesis. Consequently, genetic screening for beta-thalassemia is essential for genetic counseling, carrier screening, and prenatal diagnosis. Aim and objective This study aims to examine and identify mutations in the exon 1 region of the HBB gene in beta-thalassemia patients from the Vijayapura region. Methods This study involved 47 clinically diagnosed children with beta-thalassemia from a hospital in Vijayapura, India. Detailed clinical histories of all patients were recorded. Genomic DNA was extracted from the blood samples of these patients and subjected to polymerase chain reaction (PCR) using exon-specific primers for the HBB gene. The PCR products were then sequenced using the capillary-based Sanger sequencing method to identify mutations in the HBB gene. Results A total of 47 clinically diagnosed beta-thalassemia patients were included in the study, comprising 30 males and 17 females, aged between one and 20 years. Sequencing analysis of exon 1 in the beta-globin gene identified 17 beta-thalassemia variants. The most common mutation observed was T>G, G>C, C>A, and C>T in the exon 1 region of the HBB gene. Conclusion This study identifies the pattern of beta-thalassemia mutations, aiding in the prevention of the disorder through prenatal diagnosis and genetic counseling. Mutations can alter codon sequences, affecting protein production. Research highlights the importance of a primary prevention program to analyze mutations and sequence variations at the molecular level, thereby helping to address numerous genetic disorders.

Clinical Characteristics and Molecular Insights of Carbapenem-Resistant Klebsiella pneumoniae Isolates from Patients in Intensive Care Units.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP), a significant worldwide public health threat, is common in patients in intensive care units. Methods: A retrospective study was conducted over a period of 22 months to assess the risk factors associated with infection caused by CRKP isolates. Strain identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antimicrobial sensitivity was assessed using the micro broth dilution method and Kirby-Bauer test. The genes blaKPC, blaOXA-48, blaNDM, blaVIM, and blaGES were amplified using polymerase chain reaction (PCR), followed by sequencing of the PCR products. The polymerase hypermucoviscosity phenotype was determined using the string test. Capsular serotypes (K1, K2) and presence of the virulence gene (rmpA) in positive isolates were investigated using phenotypic tests followed by PCR. Results: Length of hospitalization and use of carbapenems were associated with CRKP infection. CRKP isolates exhibited extensive drug resistance, but retained sensitivity to colistin and ceftazidime-avibactam (CZA). The main gene detected in 35 CRKP isolates was blaKPC-2. In addition, 11 strains were positive in the string test, and two of these strains carried rmpA. Conclusions: Prolonged hospitalization and carbapenem exposure increased the risk of CRKP infection in intensive care unit (ICU) patients. The prevalence of CRKP carrying the blaKPC-2 gene was high, and suspected hypervirulent carbapenem-resistant K. pneumoniae isolates were scattered.

L-DNA-Based Melt Analysis Enables Within-Sample Validation of PCR Products.

The melt analysis feature in most real-time polymerase chain reaction (PCR) instruments is a simple method for determining if expected or unexpected products are present. High-resolution melt (HRM) analysis seeks to improve the precision of melt temperature measurements for better PCR product sequence characterization. In the area of tuberculosis (TB) drug susceptibility screening, sequencing has shown that a single base change can be sufficient to make a first-line TB drug ineffective. In this study, a reagent-based calibration strategy based on synthetic left-handed (L)-DNA, designated LHRM, was developed to confirm validation of a PCR product with single base resolution. To test this approach, a constant amount of a double-stranded L-DNA melt comparator was added to each sample and used as a within-sample melt standard. The performance of LHRM and standard HRM was used to classify PCR products as drug-susceptible or not drug-susceptible with a test bed of nine synthetic katG variants, each containing single or multiple base mutations that are known to confer resistance to the first-line TB drug isoniazid (INH). LHRM achieved comparable classification to standard HRM relying only on within-sample melt differences between L-DNA and the unknown PCR product. Using a state-of-the-art calibrated instrument and multiple sample classification analysis, standard HRM was performed at 66.7% sensitivity and 98.8% specificity. Single sample analysis incorporating L-DNA for reagent-based calibration into every sample maintained high performance at 77.8% sensitivity and 98.7% specificity. LHRM shows promise as a high-resolution single sample method for validating PCR products in applications where the expected sequence is known.

UNVEILING THE GENETIC BASIS OF BACTERIOCIN PRODUCTION FROM Enterococcus faecium ATCC 19434

The prevalence of resistance to traditional antimicrobials is increasing rapidly, leading to a significant rise in annual deaths from antibiotic-resistant illnesses. In 2020, the global death toll from such diseases was approximately 500,000. If current trends continue, this figure could exceed 10 million by 2050, with associated economic damages surpassing 100 trillion USD. These alarming statistics highlight the urgent need for alternative antimicrobials. Bacteriocins are considered among the most promising options. Enterococcus faecium ATCC 19434 is known to produce a potent bacteriocin effective against Listeria monocytogenes and Staphylococcus aureus, though its specific bacteriocin and genetic properties have not been fully explored. This study aimed to identify the gene responsible for bacteriocin production in Enterococcus faecium ATCC 19434. DNA was extracted from the bacterium, and polymerase chain reaction (PCR) was conducted. The PCR products were analyzed through agarose gel electrophoresis, sequenced, and subjected to homology searches using the BLASTN and BLASTX tools from the National Center for Biotechnology Information (NCBI). Results revealed that Enterococcus faecium ATCC 19434 contains a gene encoding enterocin B. Additionally, it was found to harbor the gene for enterocin A immunity protein production. This discovery paves the way for future genetic modification of Enterococcus faecium ATCC 19434 to enhance enterocin B synthesis, facilitating its commercial application.

Identification of an endonuclease and N6-adenine methyltransferase from Ureaplasma parvum SV3F4 strain

Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5’ extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.

Identification of novel bacterial species in the blood of patients with neonatal sepsis

BackgroundThe clinical diagnosis of neonatal sepsis remains difficult because of various challenges, such as culturing the bacteria and avoiding contamination. Therefore, this study aimed to identify bacterial pathogens in patients with clinically diagnosed neonatal sepsis by next-generation sequencing (NGS).MethodsHigh-throughput NGS and traditional culture identification were performed by comparing samples from newborns with neonatal sepsis with healthy control infants. All blood samples were separately inoculated into anaerobic and aerobic bottles and incubated for 7 days at 37 °C, the positive specimens were then identified. Novel bacteria identified through high-throughput NGS were analysed using polymerase chain reaction (PCR), PCR products were verified by Sanger sequencing. Wilcoxon rank-sum and chi-square tests were performed to assess the significance of differences in species abundance between groups. Subjects were clinically diagnosed and hospitalized at the Pediatrics Department of Shenzhen Seventh People’s Hospital and Pediatrics Department of the Longhua Branch of Shenzhen People’s Hospital. Experiments were performed at the Shenzhen Seventh People’s Hospital. The experimental group comprised 45 newborns clinically diagnosed with neonatal sepsis (age: 0–28 days; 28 males, 17 females). Fifteen normal newborns aged 0–28 days (7 males, 8 females) were included as the control group.ResultsHigh-throughput NGS showed a positivity rate of 44% (20/45) for bacteria in patients clinically diagnosed with neonatal sepsis, whereas traditional bacterial culture identification showed a positivity rate of 0% (0/45). The four main bacterial species identified were Anoxybacillus kestanbolensis, Geobacillus vulcani, Klebsiella oxytoca, and Acinetobacter guillouiae.ConclusionsA. kestanbolensis, G. vulcani, K. oxytoca, and A. guillouiae, newly discovered bacteria in patients with neonatal sepsis, were identified with high-throughput NGS. Which may result from maternal intrauterine infection or birth-canal infection and have a high clinical-cure rate. Owing to a lack of methods to culture these bacteria, their role in neonatal sepsis remains unclear. A definite diagnosis cannot rely solely on bacterial culture identification for patients with a suspected diagnosis and clinical diagnosis of neonatal sepsis and should involve other effective diagnostic techniques.

Genotype-phenotype analyses of Iranian patients with hemophilia B (Leyden -) and hemophilia B (Leyden +): A single-center study

BackgroundThere is a high prevalence of inherited bleeding disorders in Iran, such as hemophilia A (HA) and hemophilia B (HB). This study aimed to analyze the molecular and clinical profiles of patients with HB. MethodsA single-center study was conducted among patients with severe HB between March 20, 2000, and June 31, 2023. The polymerase chain reaction (PCR) amplification was used for all of the major regions, such as the promoter, the exons, the adjacent intronic regions, and the untranslated regions of the F9 gene. Finally, Sanger sequencing was performed on the PCR products. ResultsA total of 111 HB patients (17 with HB [Leyden +] and 94 with HB [Leyden -]) were enrolled in this study. Among 94 patients with HB (Leyden -), 59 (62.8 %) had missense, 21 (22.3 %) had nonsense, and 8 (8.5 %) had frameshift mutations. Moreover, the most frequent pathogenic variant in HB (Leyden +) was c.–17 A>G in this study. ConclusionThe results of this study confirm that HB is caused by a wide range of molecular defects in Iran. Thus, by knowing the genotypes and phenotypes, we would be able to stratify the patients which is important in terms of their management and outcome.

Anaplasmosis concurrent with copper deficiency in a Salem Black kid

Four months old Salem black kid was brought with the history of alopecia around the face and ear. Clinical examination revealed severe alopecia on ears, face, trunk and easy epilation of hairs along with depigmentation, mild scaling in ear margins, steely appearance, pale mucosa and loss of body condition. Detailed dermatological analysis like skin scrapings and trichogram revealed negative for ectoparasites and hair growth abnormalities. Blood smear examination revealed Anaplasma ovis organisms, with blood picture of normochromic microcytic anemia. Amplified Polymerase chain reaction product found to be positive for Anaplasma ovis. Serum biochemistry revealed low level of copper (0.0598 mg/L), elevated total bilirubin (3.6 mg/dl), direct bilirubin (2.83 mg/dl), creatinine kinase (73 mg/dl) and LDH (348 U/L). Based on above findings and treatment response, the case was diagnosed as anaplasmosis in a kid with concurrent copper deficiency.

Non-typhoidal Salmonella contributes to gastrointestinal infections in Morogoro: Evidence from patients attending Morogoro regional referral hospital in Tanzania.

Salmonella is one of the most common causes of food-borne outbreaks and infection worldwide. Non-typhoidal Salmonella (NTS) infections are common and remain a significant public health problem among important bacterial foodborne diseases. The current study aimed to establish the Non typhoidal Salmonella infection and antimicrobial resistance status among selected patients at Morogoro Regional Referral Hospital (MRRH), Morogoro Region, Tanzania, to inform clinical care management and public health interventions. A cross-sectional study was conducted using medical records and samples were collected from hospitalised and outpatients between October and December 2021. A total of 153 participants were enrolled in the study and 132 consented to being sampled. The collected samples were analysed using standard microbiological techniques. The isolates were subjected to molecular genotyping, where Polymerase Chain Reaction (PCR) was performed targeting the 16S rDNA gene. PCR products were then submitted for sequencing to establish phylogenetic relatedness. Antimicrobial susceptibility testing and resistance genes screening were also conducted. The phylogenetic analysis identified two Salmonella serovars; Salmonella Enteritidis and Salmonella Typhimurium. The isolates were from four adults and seven children patients. The isolates were tested against six antimicrobial agents: tetracycline, trimethoprim, gentamycin, ciprofloxacin, ampicillin and cefotaxime. Further antimicrobial assays were performed by screening 10 antimicrobial resistance genes using PCR. Overall, the highest resistance was observed in ampicillin (100%), whereas the lowest resistance was recorded for ciprofloxacin and gentamicin (9.1%). In addition, four (36.4%) of the isolates were resistant to cefotaxime and three (27.3%) to tetracycline and trimethoprim. The isolates also exhibit the presence of resistance genes for sulfamethoxazole 1&2, tetracycline (tet) A&B, Beta-lactamase CTXM, Beta-lactamase TEM, Beta-lactamase SHV, Gentamycine, Acra and acc3-1 in different occurrences. The overall prevalence of Salmonella species in Morogoro region was 8.3% (11/132) with Salmonella Enteritidis and Salmonella Typhimurium being the only serovars detected from adults and children stool samples. Our investigation showed that both children and adults had been exposed to Salmonella spp. However, the occurrence of NTS was higher in children (5.3% (7/132) compared to adults (3.0% (4/132). To stop zoonotic infections and the development of antimicrobial resistance in the community, this calls for Infection Prevention and Control (IPC) and stewardship programmes on rational use of antimicrobials in both health facilities and at the community level.