Epidemiological and genomic characteristics of global blaNDM-carrying Escherichia coli.

BACKGROUND: Escherichia. coli is the most frequent host for New Delhi metallo-ß-lactamase (NDM) which hydrolyzes almost all ß-lactams except aztreonam. The worldwide spread of blaNDM-carrying E. coli heavily threatens public health. OBJECTIVE: This study aimed to explore the global genomic epidemiology of blaNDM- carrying E. coli isolates, providing information for preventing the dissemination of such strains. METHODS: Global E. coli genomes were downloaded from NCBI database and blaNDM was detected using BLASTP. Per software was used to extract meta information on hosts, resources, collection data, and countries of origin from GenBank. The sequence types (STs) and distribution of antimicrobial resistance gene (ARG) were analyzed by CLC Workbench; Plasmid replicons, serotypes and virulence genes (VFs) were analyzed by submitting the genomes to the websites. Statistical analyses were performed to access the relationships among ARGs and plasmid replicons. RESULTS: Until March 2023, 1,774 out of 33,055 isolates collected during 2003-2022 were found to contain blaNDM in total. Among them, 15 blaNDM variants were found with blaNDM-5 (74.1%) being most frequent, followed by blaNDM-1 (16.6%) and blaNDM-9 (4.6%). Among the 213 ARGs identified, 27 blaCTX-M and 39 blaTEM variants were found with blaCTX-M-15 (n = 438, 24.7%) and blaTEM-1B (n = 1092, 61.6%) being the most frequent ones, respectively. In addition, 546 (30.8%) plasmids mediated ampC genes, 508 (28.6%) exogenously acquired 16 S rRNA methyltransferase encoding genes and 262 (14.8%) mcr were also detected. Among the 232 distinct STs, ST167 (17.2%) were the most prevalent. As for plasmids, more than half of isolates contained IncFII, IncFIB and IncX3. The VF terC, gad, traT and iss as well as the serotypes O101:H9 (n = 231, 13.0%), O8:H9 (n = 115, 6.5%) and O9:H30 (n = 99, 5.6%) were frequently observed. CONCLUSIONS: The study delves into the intricate relationship between plasmid types, virulence factors, and ARGs, which provides valuable insights for clinical treatment and public health interventions, and serves as a critical resource for guiding future research, surveillance, and implementation of effective strategies to address the challenges posed by blaNDM-carrying E. coli. The findings underscore the urgent need for sustained global collaboration, surveillance efforts, and antimicrobial stewardship to mitigate the impact of these highly resistant strains on public health.

Reference Intervals of Mitochondrial DNA Copy Number in Peripheral Blood for Chinese Minors and Adults.

BACKGROUND: Mitochondrial DNA (mtDNA) content measured by different techniques cannot be compared between studies, and age- and tissue-related control values are hardly available. In the present study, we aimed to establish the normal reference range of mtDNA copy number in the Chinese population. METHODS: Two healthy cohorts of 200 Chinese minors (0.1-18.0 years) and 200 adults (18.0-88.0 years) were recruited. Then, they were further categorized into eight age groups. The absolute mtDNA copy number per cell was measured by a quantitative real-time polymerase chain reaction. We subsequently used this range to evaluate mtDNA content in four patients (0.5-4.0 years) with molecularly proven mitochondrial depletion syndromes (MDSs) and 83 cases of mitochondrial disease patients harboring the m.3243A>G mutation. RESULTS: The reference range of mtDNA copy number in peripheral blood was 175-602 copies/cell (mean: 325 copies/cell) in minors and 164-500 copies/cell (mean: 287 copies/cell) in adults. There was a decreasing trend in mtDNA copy number in blood with increasing age, especially in 0-2-year-old and >50-year-old donors. The mean mtDNA copy number level among the mitochondrial disease patients with m.3243A>G mutation was significantly higher than that of healthy controls. The mtDNA content of POLG, DGUOK, TK2, and SUCLA2 genes in blood samples from MDS patients was reduced to 25%, 38%, 32%, and 24%, respectively. CONCLUSIONS: We primarily establish the reference intervals of mtDNA copy number, which might contribute to the clinical diagnosis and monitoring of mitochondrial disease.

Clinical and Molecular Characteristics in 100 Chinese Pediatric Patients with m.3243A>G Mutation in Mitochondrial DNA.

BACKGROUND: Mitochondrial diseases are a group of energy metabolic disorders with multisystem involvements. Variable clinical features present a major challenge in pediatric diagnoses. We summarized the clinical spectrum of m.3243A>G mutation in Chinese pediatric patients, to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease. METHODS: Clinical data of one-hundred pediatric patients with symptomatic mitochondrial disease harboring m.3243A>G mutation from 2007 to 2013 were retrospectively reviewed. Detection of m.3243A>G mutation ratio was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Correlation between m.3243A>G mutation ratio and age was evaluated. The differences in clinical symptom frequency of patients with low, middle, and high levels of mutation ratio were analyzed by Chi-square test. RESULTS: Sixty-six patients (66%) had suffered a delayed diagnosis for an average of 2 years. The most frequent symptoms were seizures (76%), short stature (73%), elevated plasma lactate (70%), abnormal magnetic resonance imaging/computed tomography (MRI/CT) changes (68%), vomiting (55%), decreased vision (52%), headache (50%), and muscle weakness (48%). The mutation ratio was correlated negatively with onset age (r = -0.470, P < 0.001). Myopathy was more frequent in patients with a high level of mutation ratio. However, patients with a low or middle level of m.3243A>G mutation ratio were more likely to suffer hearing loss, decreased vision, and gastrointestinal disturbance than patients with a high level of mutation ratio. CONCLUSIONS: Our study showed that half of Chinese pediatric patients with m.3243A>G mutation presented seizures, short stature, abnormal MRI/CT changes, elevated plasma lactate, vomiting, and headache. Pediatric patients with these recurrent symptoms should be considered for screening m.3243A>G mutation. Clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation.

Deletion of a 4977-bp Fragment in the Mitochondrial Genome Is Associated with Mitochondrial Disease Severity.

Large deletions in mitochondrial DNA (mtDNA) may be involved in the pathogenesis of mitochondrial disease. In this study, we investigated the relationship between a 4,977-bp deletion in the mitochondrial genome (ΔmtDNA(4977)) and the severity of clinical symptoms in patients with mitochondrial disease lacking known point mutations. A total of 160 patients with mitochondrial disease and 101 healthy controls were recruited for this study. The copy numbers of ΔmtDNA(4977) and wild-type mtDNA were determined by real-time quantitative PCR and analyzed using Spearman's bivariate correlation analysis, t-tests, or one-way ANOVA. The overall ΔmtDNA(4977) copy number per cell and the proportion of mtDNA(4977) relative to the total wild-type mtDNA, increased with patient age and symptom severity. Surprisingly, the total mtDNA copy number decreased with increasing symptom severity. Our analyses revealed that increases in the proportion and total copy number of ΔmtDNA(4977) in the blood may be associated with disease severity in patients with mitochondrial dysfunction.

[Analysis of the relationship between mitochondrial DNA deletion and clinical complexity of mitochondrial disease].

OBJECTIVE: To analyze the relationship between proportion of mitochondrial DNA 4 977 bp deletion (ΔmtDNA(4977)) or copy number in blood and the clinical complexity to find the pathogenesis of mitochondrial disease. METHODS: A total of 160 patients with mitochondrial disease and 101 healthy controls of Peking University First Hospital from December 2003 to December 2013 were collected in this study. Their peripheral blood showed no hot-point mutation which detected by polymerase chain reaction-restriction fragment length polymorphism. All the patients were divided into younger group (age<10y) and elder group (10y≤age<20y). The incidence of ΔmtDNA(4977) was detected by real-time quantitative PCR. Internal gene was used to calculate the number of mitochondrial DNA in each cell. Statistical analysis were carried out by the independent t-test, one-way ANOVA and Spearman's bivariate correlation analysis. RESULTS: ΔmtDNA (4977) proportion in the younger group was (2.66 ± 0.63)% and in the elder group was (3.09 ± 0.74)%, both of them were higher than that of healthy control group with the same age (the younger group: t=8.57, P<0.01; the elder group: t=4.38, P<0.01); ΔmtDNA(4977) copy number per cell in the younger group was (2.79 ± 0.50) copy and in the elder group was (2.97 ± 0.48) copy, both of them were higher than that of healthy control group with the same age (the younger group: t=4.50, P<0.01; the elder group: t=-3.67, P<0.01). The ΔmtDNA (4977) proportion was positively correlated with the complexity of the mitochondrial disease(the younger group: r=0.519, P<0.01; the elder group: r=0.772, P<0.01). The ΔmtDNA (4977) copy number per cell was positively correlated with the complexity of the mitochondrial disease(the younger group: r=0.389, P<0.01; the elder group: r=0.607, P<0.05). However, the total mtDNA copy number per cell was negatively correlated with the complexity of the mitochondrial disease (the younger group: r=-0.260, P<0.01; the elder group: r=-0.430, P<0.05). CONCLUSIONS: The proportion or copy number of ΔmtDNA (4977) or total mtDNA copy number in blood are correlated with the complexity of mitochondrial diseases, especially the proportion of ΔmtDNA (4977).

Trueness assessment for serum glucose measurement using commercial systems through the preparation of commutable reference materials.

BACKGROUND: Commutable reference materials (RMs) are suitable for end-users for evaluating the metrological traceability of values obtained using routine measurement systems. We assessed the performance of 6 routine measurement systems with validated secondary RMs. METHODS: We tested the homogeneity, stability, and commutability of 5 minimally processed human serum pools according to the standard guidelines. The serum pools were assigned values as per the reference procedure of the United States Centers for Disease Control and were used to evaluate the trueness of results from 6 commercial measurement systems based on enzymatic methods: 3 glucose oxidase (GOD) and 3 hexokinase (HK) methods. RESULTS: The prepared RMs were validated to be sufficiently homogenous, stable, and commutable with the patient samples. Method bias varied for different systems: GOD01, -0.17 to 2.88%; GOD02, 1.66 to 4.58%; GOD03, -0.17 to 3.14%; HK01, -3.48 to -0.85%; HK02, -3.83 to -0.11%, and HK03, -1.82 to -0.27%. CONCLUSIONS: We observed that the prepared serum glucose RMs were qualified for trueness assessment. Most of the measurement systems met the minimal quality specifications.

Application of five frozen human-pooled serum samples assigned by the International Federation of Clinical Chemistry and Laboratory Medicine reference procedure in a traceability investigation of gamma-glutamyltransferase catalytic concentration measurements in China.

BACKGROUND: The in vitro directive of the European Union requires traceability to the international recommended reference procedures. The application of the reference procedures is necessary in order to evaluate the accuracy of gamma-glutamyltransferase (GGT) assays of routine measurement systems in China. METHODS: Five frozen patient-pooled serum samples were assigned values by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure in order to evaluate the traceability of the results of GGT catalytic activity from six homogeneous systems. One of the serum samples was used to calibrate seven non-homogeneous systems. RESULTS: All of the homogeneous systems, except the Dade system (Dade Bering Inc, IL, USA), achieved traceability within the measurement range. The Roche and Hitachi systems were better than the other systems. After calibration, the variance of the non-homogeneous systems decreased dramatically from between 14.50% and 25.23% to between 1.25% and 3.09% and the bias decreased from between -11.4% and -4.1% to between 0.5% and 3.5%. CONCLUSION: Manufacturers in China should ensure that their calibration systems correspond to the IFCC reference procedures. Fresh frozen pooled patient serum assigned by reference laboratories can be used to calibrate non-homogeneous systems in order to achieve traceability.