Deciphering the genetic basis of resistome and virulome diversity among multidrug-resistant Mycoplasma hominis.

Mycoplasma hominis, a commensal bacterium that commonly inhabits the genital tract, leading to infections in both the genitourinary and extragenital regions. However, the antimicrobial resistance and pathogenic mechanisms of M. hominis isolated from extra-urogenital cystic abscess is largely unknown. This study reports the genomic epidemiological characteristics of a M. hominis isolate recovered from a pelvic abscess sample in China. Genomic DNA was extracted and sequenced using Illumina HiSeq X Ten platform. De novo assembly was performed and in silico analysis was accomplished by multiple bioinformatics tools. For phylogenomic analysis, publicly available M. hominis genomes were retrieved from NCBI GenBank database. Whole genome sequencing data showed that the genome size of M. hominis MH4246 was calculated as 679,746 bp, with 558 protein-coding sequences and a G + C content of 26.9%. M. hominis MH4246 is resistant to fluoroquinolones and macrolides, harboring mutations in the quinolone resistance-determining regions (QRDRs) (GyrA S153L, ParC S91I and ParE V417I) and 23S rRNA gene (G280A, C1500T, T1548C and T2218C). Multiple virulence determinants, such as tuf, hlyA, vaa, oppA, MHO_0730 and alr genes, were identified. Phylogenetic analysis showed that the closest relative of M. hominis MH4246 was the strain MH-1 recovered from China, which differed by 3490 SNPs. Overall, this study contributes to the comprehension of genomic characteristics, antimicrobial resistance patterns, and the mechanisms underlying the pathogenicity of this pathogen.

Ureaplasma infections: update on epidemiology, antimicrobial resistance, and pathogenesis.

Human Ureaplasma species are being increasingly recognized as opportunistic pathogens in human genitourinary tract infections, infertility, adverse pregnancy, neonatal morbidities, and other adult invasive infections. Although some general reviews have focused on the detection and clinical manifestations of Ureaplasma spp., the molecular epidemiology, antimicrobial resistance, and pathogenesis of Ureaplasma spp. have not been adequately explained. The purpose of this review is to offer valuable insights into the current understanding and future research perspectives of the molecular epidemiology, antimicrobial resistance, and pathogenesis of human Ureaplasma infections. This review summarizes the conventional culture and detection methods and the latest molecular identification technologies for Ureaplasma spp. We also reviewed the global prevalence and mechanisms of antibiotic resistance for Ureaplasma spp. Aside from regular antibiotics, novel antibiotics with outstanding in vitro antimicrobial activity against Ureaplasma spp. are described. Furthermore, we discussed the pathogenic mechanisms of Ureaplasma spp., including adhesion, proinflammatory effects, cytotoxicity, and immune escape effects, from the perspectives of pathology, related molecules, and genetics.

Global Phylogeography and Genomic Epidemiology of Carbapenem-Resistant blaOXA-232-Carrying Klebsiella pneumoniae Sequence Type 15 Lineage.

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide. To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 blaOXA-232-carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank. Phylogenetic and phylogeographic analyses revealed all blaOXA-232-carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resistance. Analysis grouped 330 global blaOXA-232-carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains. The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa. Most recent common ancestor was traced back to 2000; mutations averaged ≈1.7 per year per genome. Our research helps identify key forces driving global spread of blaOXA-232-carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP.

A multivariate analysis of the risk of iron deficiency in plateletpheresis donors based on logistic regression.

BACKGROUND: The purpose of this study was to analyze the application of individual factors, blood cell related indicators, and blood donation frequency in predicting the risk of iron deficiency of plateletpheresis donors. METHODS: A total of 801 plateletpheresis donors were included in this study. The relationship between risk factors and iron deficiency was retrospectively analyzed by univariate analysis and logistic regression analysis. The application of Hb, MCHC, RDW-CV and blood donation frequency combined prediction of iron deficiency risk among plateletpheresis donors was evaluated. RESULT: The rate of iron deficiency in this study was 31.5 % (241/766). The age, gender (the ratio of male donors), red blood cell related indicators, blood donation frequency were statistically different between the normal and iron deficiency group (all P < 0.05). Age, gender, the reciprocal of Hb and MCHC, RDW-CV, total number of blood donation and number of plateletpheresis donation in the past year, these indicators to predict the risk of iron deficiency area under the curve (AUC) were 0.558, 0.672, 0.785, 0.717, 0.599, 0.621, 0.646, respectively. The AUC of these indicators combined to predict the risk of iron deficiency was 0.877, higher than all single indicators. The sensitivity and specificity of these indicators combined in prediction of iron deficiency were 88.89 % and 81.57 %, respectively. CONCLUSION: Age, gender, the reciprocal of Hb and MCHC, RDV-CV, blood donation frequency are associated with the risk of iron deficiency in plateletpheresis donors. The combination of these indicators has high value in predicting the risk of iron deficiency.

Single-cell N6-methyladenosine regulator patterns guide intercellular communication of tumor microenvironment that contribute to colorectal cancer progression and immunotherapy.

BACKGROUND: N6-methyladenosine (m6A) RNA methylation plays a critical role in key genetic events for various cancers; yet, how m6A functions within the tumor microenvironment (TME) remains to be elucidated. METHODS: A total of 65,362 single cells from single-cell RNA-seq data derived from 33 CRC tumor samples were analyzed by nonnegative matrix factorization (NMF) for 23 m6A RNA methylation regulators. CRC and Immunotherapy cohorts from public repository were used to determine the prognosis and immune response of TME clusters. RESULTS: The fibroblasts, macrophages, T and B cells were respectively grouped into 4 to 5 subclusters and then classified according to various biological processes and different marker genes. Furthermore, it revealed that the m6A RNA methylation regulators might be significantly related to the clinical and biological features of CRC, as well as the pseudotime trajectories of main TME cell types. Bulk-seq analysis suggested that these m6A-mediated TME cell subclusters had significant prognostic value for CRC patients and distinguished immune response for patients who underwent ICB therapy, especially for the CAFs and macrophages. Notably, CellChat analysis revealed that RNA m6A methylation-associated cell subtypes of TME cells manifested diverse and extensive interaction with tumor epithelial cells. Further analysis showed that ligand-receptor pairs, including MIF - (CD74 + CXCR4), MIF - (CD74 + CD44), MDK-NCL and LGALS9 - CD45, etc. mediated the communication between m6A associated subtypes of TME cells and tumor epithelial cells. CONCLUSIONS: Taken together, our study firstly revealed the m6A methylation mediated intercellular communication of the tumor microenvironment in the regulation of tumor growth and antitumor immunomodulatory processes.

Development of nomograms to predict therapeutic response and prognosis of non-small cell lung cancer patients treated with anti-PD-1 antibody.

BACKGROUND: Anti-programmed death-1 (PD-1) antibody changed the treatment of non-small cell lung cancer (NSCLC), however, reliable predictive markers were lacking. We aimed to explore factors associated with response and survival, and develop predictive models. METHODS: This multicenter retrospective study included a training cohort (n = 92) and validation cohort (n = 111) with NSCLC patients received anti-PD-1 antibody monotherapy in eight Chinese hospitals, and a control cohort (n = 124) with NSCLC patients received chemotherapy. Logistic and Cox models were used to identify factors associated with response and survival respectively. Nomograms were developed based on significant factors, and evaluated by Concordance-index (C-index), area under the curve (AUC) and calibration curve. RESULT: In training cohort, smoking history (P = 0.027) and higher absolute lymphocyte count (P = 0.038) were associated with response. Female (P < 0.001), age ≥ 65 years (P = 0.004) and higher lactate dehydrogenase (LDH, P < 0.001) were associated with shorter progression-free survival (PFS). Higher LDH (P < 0.001) and derived neutrophil-to-lymphocyte ratio (P = 0.035) were associated with poorer overall survival (OS). While these factors were nonsignificant in chemotherapy cohort. Three nomograms to predict response at 6-week after treatment, PFS and OS at 6-, 12- and 18-months were developed, and validated in validation cohort. The C-indices of each nomogram in both cohorts were as follow (training vs validation): 0.706 vs 0.701; 0.728 vs 0.701; 0.741 vs 0.709; respectively. AUC showed a good discriminative ability. Calibration curves demonstrated a consistence between actual results and predictions. CONCLUSION: We developed predictive nomograms based on easily available factors to help clinicians early assess response and prognosis for NSCLC patients received anti-PD-1 antibody.

Mechanism evaluation for an amino acid substitution p.Y246C of B-glycosyltransferase enzyme with Bweak phenotype.

BACKGROUND: The amino acid substitutions caused by ABO gene variants are usually predicted to impact the glycosyltransferase function. Here, the effect of an amino acid substitution in the vicinity of the catalytic active region of the B-glycosyltransferase was explored in vitro and in silico study, which is important for further recognizing the ABO subgroup. METHODS: The ABO serological tests were performed by the routine methods. The ABO genotype was analyzed by polymerase chain reaction and sequenced bidirectionally. The haplotype of the variant allele was separated using single-strand amplification and sequencing with allele-specific primers. Stably expression cell lines with variant were constructed for study in vitro. 3D structure of the B-glycosyltransferase (GTB) variant was simulated by PyMOL software. The free energy change (ΔΔG) was calculated by FoldX. RESULTS: A variant c.737A > G was identified in a Chinese individual with Bweak phenotype, which led to an amino acid substitution p.Y246C in the vicinity of the catalytic active region of GTB enzyme. The stably expression cell lines with variant and wild type were successfully established and showed that the variant caused a decrease in protein levels and/or enzyme activity. The 3D structural of the GTB modelling found the amino acid substitution p.Y246C caused the hydrogen bond of the protein changes. Meanwhile, the free energy change (ΔΔG) value predicted the destabilizing effect on the variant GTB. DISCUSSION: The p.Y246C variant in the vicinity of the enzyme active centre reduced the antigen expression because of greatly destabilizing effect on the GTB variant.

Antimicrobial Resistance in Clinical Ureaplasma spp. and Mycoplasma hominis and Structural Mechanisms Underlying Quinolone Resistance.

Antibiotic resistance is a global concern; however, data on antibiotic-resistant Ureaplasma spp. and Mycoplasma hominis are limited in comparison to similar data on other microbes. A total of 492 Ureaplasma spp. and 13 M. hominis strains obtained in Hangzhou, China, in 2018 were subjected to antimicrobial susceptibility testing for levofloxacin, moxifloxacin, erythromycin, clindamycin, and doxycycline using the broth microdilution method. The mechanisms underlying quinolone and macrolide resistance were determined. Meanwhile, a model of the topoisomerase IV complex bound to levofloxacin in wild-type Ureaplasma spp. was built to study the quinolone resistance mutations. For Ureaplasma spp., the levofloxacin, moxifloxacin, and erythromycin resistance rates were 84.69%, 51.44%, and 3.59% in U. parvum and 82.43%, 62.16%, and 5.40% in U. urealyticum, respectively. Of the 13 M. hominis strains, 11 were resistant to both levofloxacin and moxifloxacin, and five strains showed clindamycin resistance. ParC S83L was the most prevalent mutation in levofloxacin-resistant Ureaplasma strains, followed by ParE R448K. The two mutations GyrA S153L and ParC S91I were commonly identified in quinolone-resistant M. hominis A molecular dynamics-refined structure revealed that quinolone resistance-associated mutations inhibited the interaction and reduced affinity with gyrase or topoisomerase IV and quinolones. The novel mutations S21A in the L4 protein and G2654T and T2245C in 23S rRNA and the ermB gene were identified in erythromycin-resistant Ureaplasma spp. As fluoroquinolone resistance in Ureaplasma spp. and Mycoplasma hominis remains high in China, the rational use of antibiotics needs to be further enhanced.

Nicotinamide N-methyltransferase inhibits autophagy induced by oxidative stress through suppressing the AMPK pathway in breast cancer cells.

BACKGROUND: Nicotinamide N-methyltransferase (NNMT) is highly expressed in several cancers and can regulate cell epigenetic status and various cell metabolism pathways, such as ATP synthesis and cellular stress response. We reported in our previous papers that NNMT overexpression inhibits the apoptosis and enhances the chemotherapy resistance of breast cancer cells. This study aims to investigate the effect of NNMT on autophagy induced by oxidative stress in breast cancer cells, which might provide a novel therapeutic strategy for breast cancer treatment. METHODS: NNMT and LC3B II protein levels in the two cell models (SK-BR-3 and MDA-MB-231) with NNMT overexpression or knockdown were detected by Western blotting and correlated with each other. Changes in cellular viability, intracellular reactive oxygen species (ROS) and ATP levels were assessed after H2O2 treatment. Then, autophagosomes were imaged by transmission electron microscopy, and LC3 puncta were examined by confocal microscopy and flow cytometry. The LC3B II level and AMPK-ULK1 pathway activity were both detected by Western blotting to determine the role of NNMT in the H2O2-induced autophagy. RESULTS: NNMT expression was negatively correlated with LC3B II expression in both cell models (SK-BR-3 and MDA-MB-231). Then, NNMT overexpression attenuated the autophagy induced by H2O2 in SK-BR-3 cells, whereas knockdown promoted autophagy induced by H2O2 in MDA-MB-231 cells. Furthermore, mechanistic studies showed that NNMT suppressed the ROS increase, ATP decrease and AMPK-ULK1 pathway activation, resulting in the inhibition of H2O2-induced autophagy in breast cancer cells. CONCLUSIONS: We conclude that NNMT inhibits the autophagy induced by oxidative stress through the ROS-mediated AMPK-ULK1 pathway in breast cancer cells and may protect breast cancer cells against oxidative stress through autophagy suppression.

Combining serum calcitonin, carcinoembryonic antigen, and neuron-specific enolase to predict lateral lymph node metastasis in medullary thyroid carcinoma.

BACKGROUND: This study aimed to investigate the clinical application of combined detection of serum calcitonin (Ctn), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE) in predicting lateral lymph node metastasis (LLNM) in medullary thyroid carcinoma (MTC). METHODS: Seventy-four consecutive patients with MTC were enrolled. The relationship between serum Ctn, CEA, and NSE and LLNM was retrospectively analyzed by univariate analysis and logistic regression analysis. Furthermore, the clinical application of serum Ctn, CEA, and NSE combined detection in prediction of LLNM in MTC was also evaluated. RESULTS: The rate of LLNM in this study was 48.64% (36/74).The expression levels of serum Ctn, CEA, and NSE in MTC with LLNM were significantly higher than those without LLNM (all P < .01). The area under the curve (AUC) predicted by serum Ctn, CEA, and NSE for LLNM in MTC patients was 0.867, 0.831, and 0.726, respectively, and the AUC of serum Ctn, CEA, and NSE combined detection was up to 0.890, higher than using a single biomarker. The sensitivity and specificity of serum Ctn, CEA, and NSE combined detection in prediction of LLNM were 88.89% and 81.57%, respectively. CONCLUSIONS: The concentrations of serum Ctn, CEA, and NSE are closely related to LLNM in MTC, and the combined detection of all three biomarkers has a higher clinical value in the evaluation of MTC patients with LLNM. With more perspective study in the future, it would be an indicator of influencing personalized surgical strategy for different MTC patients.

Molecular Basis of ABO Variants Including Identification of 16 Novel ABO Subgroup Alleles in Chinese Han Population.

INTRODUCTION: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion. METHODS: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software. RESULTS: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO*A alleles, 23 ABO*B alleles, 4 ABO*BA alleles, 4 ABO*cisAB alleles, and 1 ABO*O alleles. ABO*A2.08 and ABO*BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression. CONCLUSIONS: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety.

Nicotinamide N-methyltransferase enhances chemoresistance in breast cancer through SIRT1 protein stabilization.

BACKGROUND: Nicotinamide N-methyltransferase (NNMT) is overexpressed in various human tumors and involved in the development and progression of several carcinomas. In breast cancer, NNMT was found to be overexpressed in several cell lines. However, the clinical relevance of NNMT in breast cancer is not yet clear. METHODS: NNMT expression in breast carcinoma was examined by immunohistochemistry, and then, its relationship with patient clinicopathological characteristics was analyzed. The effects of NNMT on chemoresistance in breast cancer cells were assessed by cell viability, colony formation, and apoptosis assay. The NNMT, SIRT1, p53, and acetyl-p53 proteins, which are involved in NNMT-related chemoresistance, were examined by Western blotting. The SIRT1 mRNA was examined by real-time PCR, and its activity was measured by using the SIRT1 deacetylase fluorometric reagent kit. RESULTS: NNMT expression was significantly higher (53.9%) in breast carcinoma than in paracancerous tissues (10.0%) and breast hyperplasia (13.3%). A high level of NNMT expression correlated with poor survival and chemotherapy response in breast cancer patients who received chemotherapy. Ectopic overexpression of NNMT significantly inhibited the apoptotic cell death and suppression of colony formation induced by adriamycin and paclitaxel. Mechanistic studies revealed that NNMT overexpression increased SIRT1 expression and promoted its activity. Either inhibition of SIRT1 by EX527 or knockdown of SIRT1 by siRNA could reverse NNMT-mediated resistance to adriamycin and paclitaxel, which suggests that SIRT1 plays a critical role in NNMT-related chemoresistance in breast cancer. CONCLUSIONS: The results of this study demonstrate a novel correlation between the NNMT expression level and patient survival, suggesting that NNMT has the potential to become a new prognostic biomarker to predict the treatment outcomes of the clinical chemotherapy in breast cancer. Moreover, targeting NNMT or downstream SIRT1 may represent a new therapeutic approach to improve the efficacy of breast cancer chemotherapy.

Emerging challenges of whole-genome-sequencing-powered epidemiological surveillance of globally distributed clonal groups of bacterial infections, giving Acinetobacter baumannii ST195 as an example.

Whole-genome sequencing (WGS) has revolutionized the genotyping of bacterial pathogens and is expected to become the new gold standard for tracing the transmissions of bacterial infectious diseases for public health purposes. However, it is still unexpectedly demanding to employ WGS for global epidemiological surveillance because of the high degree of similarity between the genomes of intercontinental isolates. The aim of this study was to utilize genomically derived bioinformatics analysis to identify globally distributed A. baumannii ST195 lineage and differentiation outbreaks to address this issue. The genomic sequences and their related epidemiological metadata of 2850 A. baumannii isolates were recruited from NCBI Genbank database. Assignment into sequence type (Oxford scheme) and lineage (global clone 2/CC92) were performed. A total of 91 ST195 A. baumannii isolates were subsequently classified to perform the bacterial source tracking analysis by implementing both core genome MLST (cgMLST) and core genome SNP (cgSNP) strategy that were integrated in our recently updated BacWGSTdb 2.0 server. Antibiotic resistance genes were identified using the ResFinder database. The ST195 A. baumannii isolates distributed widely in eight countries and harboured multiple antimicrobial resistance genes simultaneously. In most cases, the bacterial isolates recovered from geographically distant sources may present less genomic sequence similarity, i.e., the phylogenetic relationship between these ST195 isolates worldwide was roughly congruent with their country of isolation. However, a few isolates collected from distant geographic regions were revealed to possess smaller genetic distances (less than 8 loci or 20 SNPs) than the threshold without an observable epidemiological link. Our study highlights the emerging challenges entailed in the WGS-powered epidemiological surveillance of globally distributed clonal groups. Standardization is urgently required before WGS can be routinely applied to infectious diseases outbreak investigations.

Ureaplasma spp. lipid-associated membrane proteins induce human monocyte U937 cell cycle arrest through p53-independent p21 pathway.

Ureaplasma spp. are known to be associated with human genitourinary tract diseases and perinatal diseases and Ureaplasma spp. Lipid-associated membrane proteins (LAMPs) play important roles in their related diseases. However, the exact mechanism underlying pathogenesis of Ureaplasma spp. LAMPs is largely unknown. In this study, we explored the pathogenic mechanisms of Ureaplasma spp. LAMPs by elucidating their role in modulating the cell cycle and related signaling pathways in human monocytic cell U937, which is highly related to the inflammatory and protective effect in infectious diseases. We utilized the two ATCC reference strains (Ureaplasma parvum serovar 3 str. ATCC 27,815 (UPA3) and Ureaplasma urealyticum serovar 8 str. ATCC 27,618 (UUR8)) and nine clinical isolates which including both UPA and UUR to study the effects of Ureaplasma spp. LAMPs on U937 in vitro. We found that LAMPs derived from UUR8 and both UPA and UUR of clinical strains markedly inhibited the cell proliferation, while UPA3 LAMPs suppressed slightly. Besides, the result of flow cytometry analysis indicated all the Ureaplasma spp. LAMPs could arrest U937 cells in G1 phase. Next, we found that the cell cycle arrest was associated with increased levels of p53 and p21, and a concomitant decrease in the levels of CDK2, CDK4, CDK6 and cyclin E1 at both transcriptional and translational levels after treatment with LAMPs derived from UUR8 or clinical strains, while only cyclin E1 was down-regulated after treatment with UPA3 LAMPs. Further study showed that p53 down-regulation had almost no effect on the distribution of cell cycle and the expression of p21. In conclusion, this study demonstrated that LAMPs derived from UUR8 and clinical strains could inhibit the proliferation of U937 cells by inducing G1 cell cycle arrest through increasing the p21 expression in a p53-independent manner, while UPA3 LAMPs could induce the cell cycle arrest slightly. Our study could contribute to the understanding of Ureaplasma spp. pathogenesis, which has potential value for the treatment of Ureaplasma spp. infections.

Clonal diversity of Ureaplasma species and its relationship with oligozoospermia and semen quality in Chinese infertile males.

Whether Ureaplasma spp. are a causative agent of male infertility remains controversial. Previous studies concerning Ureaplasma spp. and male infertility have been confined to the species level of Ureaplasma. Currently, an expanded multilocus sequence typing (eMLST) scheme has been established with high discriminatory power. The aim of this study was to use eMLST to explore the distribution of Ureaplasma spp. and to analyze its role in oligozoospermia and semen quality. A total of 480 semen samples were obtained from Chinese infertile males. The associations between Ureaplasma spp. with oligozoospermia and semen characteristics were further evaluated. Phylogenetic analysis revealed that 102 Ureaplasma spp. could be separated into two clusters and seven sub-groups. Within cluster I (U. parvum), eST16 and eST41 were the most frequent clones. For cluster II (U. urealyticum), eST82 and eST147 were the most prevalent clones. Sub-groups A and C belonging to cluster I and sub-group 1 belonging to cluster II showed an association with oligozoospermia, in contrast with the Ureaplasma spp. negative group (P < 0.05). Compared with the negative group, semen motility decreased in sub-group 2, especially for non-progressive motility (P < 0.05). These results indicated that sub-groups A and C belonging to cluster I (U. parvum) and sub-group 1 belonging to cluster II (U. urealyticum) were shown to be associated with oligozoospermia. Sub-group 2 belonging to cluster II may have the ability to impair semen motility, especially for non-progressive motility.

Reference intervals for biochemical, haemostatic and haematological parameters in healthy Chinese women during early and late pregnancy.

BACKGROUND: Variations in maternal blood parameters, which are mostly induced by the physiological changes that occur during pregnancy, have been reported in different gestational periods. The use of the established reference intervals for healthy adult females leads to the misclassification of healthy pregnant women as abnormal. Our aim was to establish appropriate reference intervals for biochemical, haematological and haemostatic parameters in the first and third trimesters of pregnancy. METHODS: We included 565 healthy pregnant women with normal pregnancies. Blood samples were collected for biochemical analyses, complete blood counts and coagulation analyses at 8-12 and 28-37 weeks of gestation. The median and reference intervals (the 2.5th and 97.5th values) were calculated for each parameter during pregnancy and then compared to the established reference intervals for healthy adult females. RESULTS: Significant increases in triglyceride, total cholesterol, low-density lipoprotein cholesterol, uric acid, alkaline phosphatase, white blood cell, mean platelet volume, fibrinogen and D-dimer reference intervals and clear decreases in total protein, albumin, blood urea nitrogen, creatinine, red blood cell, haemoglobin, haematocrit, platelet counts and thrombin time reference intervals were observed during pregnancy. According to the 'n%', most changes were observed beginning in the first trimester. Compared to the established reference intervals, the greatest misclassifications were observed for ALB, ALP and D-Di. CONCLUSIONS: Changes in maternal blood parameters during pregnancy were confirmed. We recommend that the reference intervals for most blood parameters be revised to account for the gestational period.

Measurement of HE4 and CA125 and establishment of reference intervals for the ROMA index in the sera of pregnant women.

INTRODUCTION: Cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) are biomarkers for ovarian cancer. Their specificity and sensitivity are often limited during pregnancy as a result of great fluctuations. The risk of ovarian malignancy algorithm (ROMA) score, which combines CA125, HE4, and menopausal status, may improve diagnostic performance. There are no reports regarding the ROMA index in pregnant women. Therefore, the aim of our study was to establish appropriate reference intervals (RIs) for the ROMA index in pregnant Chinese women and compare them with those of CA125 and HE4 during pregnancy. METHODS: Serum concentrations of CA125 and HE4 were simultaneously measured in healthy pregnant women via electrochemiluminescence immunoassay (ECLIA). The ROMA index was calculated using premenopausal algorithms. RESULTS: The RIs for the ROMA index calculated by premenopausal algorithms were substantially closer to the normal range in the first 2 trimesters. For pregnant women, the great misclassifications identified in CA125 may be reversed by the use of ROMA index. CONCLUSIONS: We established the RIs for HE4 and CA125, as well as the ROMA index, in pregnant women at different gestational periods. The ROMA index is suggested to be a more promising tumor marker for pregnant women diagnosed with malignance.

Detection of an Escherichia coli Sequence Type 167 Strain with Two Tandem Copies of blaNDM-1 in the Chromosome.

New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the blaNDM-1 gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant Escherichia coli strain Y5, originating from a urine sample and containing the blaNDM-1 gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of blaNDM-1, the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with Escherichia coli 6409 (GenBank accession no. CP010371), which lacks the same blaNDM-1 gene-surrounding structure as Y5. The blaNDM-1 gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (ISCR1) element (sul1-ARR-3-cat-blaNDM-1-bleo-ISCR1), which appears intact in the plasmid from Proteus mirabilis (GenBank accession no. KP662515). The genomic context indicates that the ISCR1 element mediated the blaNDM-1 transposition from a single source plasmid to the chromosome. Our study is the first report of an Enterobacteriaceae strain harboring a chromosomally integrated blaNDM-1, which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor blaNDM-1.

Performance of the Sysmex UF-1000i urine analyser in the rapid diagnosis of urinary tract infections in hospitalized patients.

BACKGROUND: Urinary tract infections (UTIs) are the second most frequently encountered nosocomial infectious diseases, and they greatly increase the cost of medical care and prolong the duration of hospital stays. AIM: We aimed to evaluate the performance of the Sysmex UF-1000i analyser for the rapid prediction of UTIs in hospitalized patients with or without indwelling catheters at a comprehensive teaching hospital that encounters complex disease types. METHODS: Urine specimens (n = 1016) were cultured and examined for WBC, RBC, bacteria (BACT) and yeast-like cell (YLC) count using the Sysmex UF-1000i. The results were compared with the urine culture results. Receiver operating characteristic curve analysis was applied to determine BACT and YLC cutoff values for bacterial and fungal UTIs independently. The diagnostic ability of the UF-1000i was also compared for patients with and without indwelling catheters. FINDINGS: A cutoff value of 38.7/µL was acceptable for ruling out bacterial UTIs. In this setting, we achieved a sensitivity of 90%, a negative predictive value of 94.5%, a false negative rate of 2.85% (29 cases), and avoided culturing in 52% of the samples. The BACT count presented a larger area under the curve for patients with indwelling catheters than for those without (0.939 vs. 0.861); however, no significant difference in the diagnostic ability of the two curves was found. CONCLUSION: The Sysmex UF-1000i analyser could be a reliable method for excluding bacterial UTIs in hospitalized patients with or without urinary catheters and could help clinicians determine whether antibiotic therapy is necessary.

[Expression of microRNA-221/222 in patients with monoclonal gammopathy of undetermined significance and multiple myeloma].

Objective: To detect the expression of miR-221/222 in serum and plasma cells in patients with monoclonal gammopathy of undetermined significance(MGUS) and multiple myeloma(MM), and to explore the possibility of miR-221/222 as biomarkers in the diagnosis and prognosis predicting of MGUS and MM. Methods: Bone marrow and serum samples from 14 patients with newly diagnosed MGUS, 81 patients with newly diagnosed or relapsed MM and 10 controls were collected from Sir Run Run Shaw Hospital of Zhejiang University and Tongde Hospital of Zhejiang Province during January 2013 and December 2015. The expressions of miR-221/222 in serum and in sorted CD138 positive plasma cells were detected by qRT-PCR, and the relative expression of miR-221/222 (Δct) was compared between the groups. Serum levels of miR-221 before and after treatment were compared in both remission group (n=22) and refractory group (n=13) in MM patients, and its correlation with serum level of ß2-MG was assessed using Pearson's correlation analysis. Results: Serum levels of miR-221/222 in MGUS and MM groups were significantly higher than those in control group (all P<0.01), while miR-221/222 levels in plasma cells were significantly lower in MGUS and MM groups than those in the control group (P<0.05 or<0.01). No significant difference in miR-221/222 levels in serum and plasma cells was observed between MGUS group and MM group (all P>0.05). There was no correlation between miR-221/222 levels in serum and plasma cells (r=0.024 and -0.127, all P>0.05), but miR-221 levels were correlated with miR-222 levels in both serum and plasma cells (r=0.534 and 0.552, all P<0.01). Receiver operating characteristic (ROC) curves showed that the areas under the curve (AUCs) of serum miR-221/222, plasma cell miR-221/222 in diagnosis of MGUS/MM were 0.968, 0.976, 0.801 and 0.727, respectively. There was no significant difference in serum level of miR-221 among MM patients with different paraprotein isotypes (P>0.05), but serum level of miR-221 in patients with relapsed MM was higher than that in patients with newly diagnosed MM (P<0.01). Compared with the patients with MGUS or MM stageⅠ and Ⅱ, patients with MM stage Ⅲ were of higher serum levels of miR-221 (P<0.01). Serum level of miR-221 decreased after chemotherapy in the remission group (U=51.5, P<0.01), but such decrease was not observed in the refractory group (U=67.5, P>0.05). Serum level of ß2-MG was positively correlated with serum level of miR-221 (r=0.524, P<0.01). Conclusion: miR-221/222 in serum and plasma cells may be biomarkers for early diagnosis of MGUS, and are helpful for diagnosis and efficacy evaluation of MM.