Comparison of Cerebrospinal Fluid Cellular Analysis by Optical Microscopy and Sysmex XN 9000.

BACKGROUND: This study was designed to compare the body fluid module of Sysmex XN9000 (XN-BF) with optical microscopy (OM) for cerebrospinal fluid (CSF) analysis after two-step cell slide centrifuge (TSCSC), defining the best procedure for CSF optical microscopy analysis. METHODS: Items of RBC, WBC enumeration and differentiation were observed. The cell count and morphologic evaluation of the cellular composition by OM was carried out both with and without two-step cell slide centrifuge (TSCSC) and were compared the data with XN-BF. RESULTS: There were 69.98 ± 4.94 RBC and 36.98 ± 3.39 WBC in one OSCSC microscopic field whereas there were 96.35 ± 5.41 RBC and 66.15 ± 4.85 WBC in one TSCSC microscopic field in the same sample (*200). There was a statistical difference between those two methods (p = 0.000). Excellent correlation was found between total cell count with both OM and XN-BF. The R2 value for RBC and WBC counts were 0.99 and 0.96, respectively. For WBC differential, the R2 values were 0.98 for PMN and 0.70 for MN. Correlation of MN was poorer than PMN. As far as the tumor cell, phagocyte, and plasma cell with high fluorescence were concerned, OM were not consistent with XN-BF. CONCLUSIONS: The TSCSC procedure contributes to the separation of cells and other ingredients. XN-BF displays excellent performance at RBC and WBC cell count except for mononuclear cells, tumor cells, phagocytes, and leukemia cells. which makes it just a practical alternative to total cell (WBC, RBC) count for CSF samples. Detailed morphologic workup of CSF samples is mandated in all cases with meningoencephalitis, elevated cell count, sub-arachnoid hemorrhage and meningeal carcinomatosis, the TSCSC procedure is recommended.

Identification of Peptoniphilus vaginalis-Like Bacteria, Peptoniphilus septimus sp. nov., From Blood Cultures in a Cervical Cancer Patient Receiving Chemotherapy: Case and Implications.

A 39-year-old woman with a 3-year human papillomavirus (HPV) 18 infection history was admitted to the hospital for a 16-day history of vaginal bleeding after sex. She was diagnosed with cervical cancer based on the results of the electronic colposcopy, cervical cytology, microscopy, and magnetic resonance imaging (MRI). Then, she received chemotherapy, with paclitaxel 200 mg (day 1), cisplatin 75 mg (day 2), and bevacizumab 700 mg (day 3) twice with an interval of 27 days. During the examination for the diagnosis and treatment, many invasive operations, including removal of intrauterine device, colposcopy, and ureteral dilatation, were done. After that, the patient was discharged and entered the emergency department about 2.5 months later with a loss of consciousness probably caused by septic shock. The patient finally died of multiple organ failure and bacterial infection, although she has received antimicrobial therapy. The blood cultures showed a monobacterial infection with an anaerobic Gram-positive bacterial strain, designated as SAHP1. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the patient was infected with Peptoniphilus asaccharolyticus, while molecular analysis and genome-based taxonomy confirmed the infection with a novel Peptoniphilus species that has a close genetic relationship with Peptoniphilus vaginalis and proposed provisionally as Peptoniphilus septimus sp. nov., which may also act as a commensal of the human vagina. Genomic features of SAHP1 have been fully described, and comparative genomic analysis reveals the known prokaryote relative of Peptoniphilus septimus sp. nov. in the genus Peptoniphilus. The invasive operations on the genital tract during the diagnosis and treatment of the patient and the tumor tissue damage and bleeding may have a certain role in the bloodstream infection. This study casts a new light on the Peptoniphilus bacteria and prompts clinicians to include anaerobic blood cultures as part of their blood culture procedures, especially on patients with genital tract tumors. Furthermore, due to the incomplete database and unsatisfying resolution of the MALDI-TOF MS for Peptoniphilus species identification, molecular identification, especially whole-genome sequencing, is required for those initially identified as bacteria belonging to Peptoniphilus in the clinical laboratory.

A Modified Ink Staining Procedure for Cryptococcus Detection of the Cerebrospinal Fluid by Microscope.

BACKGROUND: The pathogen detection rate of traditional ink staining procedure (TISP) is low. METHODS: A modified ink staining procedure (MISP) was created to accumulate pathogen by using cell slide centrifuge. Items of RBC, WBC, and cryptococcus were observed. RESULTS: There were 79.98 ± 54.94 RBC and 126.98 ± 36.39 WBC in one MISP microscopic field whereas there were only 3.35 ± 2.41 RBC and 6.15 ± 1.85 WBC in one TISP microscopic field in the same sample (*200). There was statistical difference between those two methods (p = 0.000). There were 40.78 ± 13.23 mL cryptococcus in CSF processed by MISP whereas there were only 2.10 ± 1.10 mL cryptococcus in the same CSF processed by TISP. There was statistical difference between those two methods. CONCLUSIONS: The modified ink staining procedure contributes to the separation of cells and pathogens (such as cryptococcus).

Virulence effector SidJ evolution in Legionella pneumophila is driven by positive selection and intragenic recombination.

Effector proteins translocated by the Dot/Icm type IV secretion system determine the virulence of Legionella pneumophila (L. pneumophila). Among these effectors, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism mediated by another effector, SidJ. Host-cell calmodulin (CaM) activates SidJ to glutamylate the SidEs of ubiquitin (Ub) ligases and to make a balanced Ub ligase activity. Given the central role of SidJ in this regulatory process, studying the nature of evolution of sidJ is important to understand the virulence of L. pneumophila and the interaction between the bacteria and its hosts. By studying sidJ from a large number of L. pneumophila strains and using various molecular evolution algorithms, we demonstrated that intragenic recombination drove the evolution of sidJ and contributed to sidJ diversification. Additionally, we showed that four codons of sidJ which are located in the N-terminal (NTD) (codons 58 and 200) and C-terminal (CTD) (codons 868 and 869) domains, but not in the kinase domain (KD) had been subjected to strong positive selection pressure, and variable mutation profiles of these codons were identified. Protein structural modeling of SidJ provided possible explanations for these mutations. Codons 868 and 869 mutations might engage in regulating the interactions of SidJ with CaM through hydrogen bonds and affect the CaM docking to SidJ. Mutation in codon 58 of SidJ might affect the distribution of main-chain atoms that are associated with the interaction with CaM. In contrast, mutations in codon 200 might influence the α-helix stability in the NTD. These mutations might be important to balance Ub ligase activity for different L. pneumophila hosts. This study first reported that intragenic recombination and positive Darwinian selection both shaped the genetic plasticity of sidJ, contributing to a deeper understanding of the adaptive mechanisms of this intracellular bacterium to different hosts.

[Effect of acupuncture on pain-emotion related brain regions in patients with cervical spondylosis of cervical type: a fMRI study].

OBJECTIVE: To observe the changes of functional connectivity of brain pain-emotion regulation region in patients with cervical spondylosis of cervical type by functional magnetic resonance imaging (fMRI). METHODS: Thirty-two subjects were selected. Of them, 16 patients with cervical spondylosis of cervical type were divided into an observation group and 16 healthy subjects into a control group. The patients in the observation group were treated with acupuncture at Tianzhu (BL 10), Jingbailao (EX-HN 15), Jianzhongshu (SI 15) and ashi points for 30 min. The rest-state fMRI data was collected before and after acupuncture in the observation group. The subjects in the control group received no treatment, and the rest-state fMRI data was collected once. The visual analogue scale (VAS) score before and after treatment and the pain catastrophizing scale (PCS) score before treatment in the observation group were recorded. The resting-state brain functional imaging characteristics between the observation group and control group before treatment, between the observation group before and after treatment, were compared. Based on the brain functional connectivity of region of interest (ROI) the changes of functional connectivity in insula and ventral tegmental area (VTA) in emotional regulation brain region were observed, and the correlation between functional connectivity changes and VAS、PCS scores in patients of the observation group was analyzed. RESULTS: In the observation group, the VAS score was (1.94±1.12) after the treatment, which was lower than (5.62±1.20) before treatment (P<0.05). The PCS score before treatment was (19.18±8.42) in the observation group. Compared with the control group, the areas with increased functional connectivity with insula in the observation group before acupuncture included bilateral dorsolateral prefrontal lobe and right middle cingulate gyrus, and the areas with increased functional connectivity with VTA included right central posterior gyrus and right insula. In the observation group, the connectivity coefficient of left insula and left dorsolateral prefrontal lobe (r=0.438, P<0.05), the connectivity coefficient of right insula and right dorsolateral prefrontal lobe (r=0.483, P<0.05) were positively associated with the VAS score. In the observation group, the connectivity coefficient between the right insula and the right middle cingulate gyrus (r=-0.560, P<0.05), the connectivity coefficient between the right VTA and the right insula (r=-0.525, P<0.05) were negatively associated with the PCS score. After acupuncture, the areas with decreased functional connectivity with insula included bilateral posterior central gyrus, right anterior central gyrus, middle cingulate gyrus and left corpus callosum, while the bilateral suboccipital gyrus and left cerebellum showed increased functional connectivity with right insula. The areas with decreased functional connectivity with VTA included bilateral dorsomedial prefrontal cortex, left anterior cingulate gyrus, right middle temporal gyrus and left anterior cingulate gyrus. After acupuncture in the observation group, the functional connectivity of left VTA left dorsomedial prefrontal cortex and left anterior cingulate cortex (r=-0.548, P<0.05), the functional connectivity of right VTA-bilateral dorsomedial prefrontal cortex and left anterior cingulate cortex (r=-0.547, P<0.05) were negatively associated with the PCS score. CONCLUSION: Pain involves the formation and expression of "pain-emotion-cognition". Acupuncture can systematically regulate the brain functional connections between cognitive regions such as dorsal prefrontal lobe and anterior cingulate gyrus and emotional regions such as insula and VTA in patients with cervical spondylosis of cervical type, suggesting that acupuncture has a multi-dimensional and comprehensive regulation effect on pain.

Adjuvant concomitant treatment with traditional Chinese medicines in patients receiving chemotherapy for HER2-Positive breast cancer: A pilot randomized controlled trial.

This study aimed to evaluate the impact of Chinese medicine on controlling cancer and easing adverse events in patients with HER2-positive breast cancer. We recruited consecutive HER2-positive breast cancer patients who underwent radical mastectomy from January 2015 to January 2019. Patients were randomly assigned to receive chemotherapy plus Chinese medicine or chemotherapy alone. The left ventricular global longitudinal strain was better in the experimental group (P < 0.01). The reduction in white blood cells was more significant in the control group (P < 0.01). Hepatic function in the experimental group was better than that in control group after chemotherapy (P < 0.01). In addition, the scores of symptom dimensions for pain, diarrhea, and hair loss were better in the experimental group than in the control group after chemotherapy (P < 0.01). For patients with HER2-positive breast cancer, personalization of traditional Chinese medicine can not only enhance the anti-cancer function of chemotherapy but also ease serious adverse effects.

Up-regulated microRNA-199b-3p represses the apoptosis of cerebral microvascular endothelial cells in ischemic stroke through down-regulation of MAPK/ERK/EGR1 axis.

MicroRNAs (miRNAs) have emerged as key mediators of posttranscriptional gene silencing in both pathogenic and pathological aspects of ischemic stroke biology. Therefore, the purpose of present study was to explore the effect of microRNA-199b-3p (miR-199b-3p) on the cerebral microvascular endothelial cells (CMECs) in middle cerebral artery occlusion-reperfusion (MCAO-R) mice by regulating MAPK/ERK/EGR1 axis. Mice were used to establish MCAO-R models and to measure the expression of miR-199b-3p and the MAPK/ERK/EGR1 axis-related genes. CMECs were extracted from the MCAO-R mice. A series of mimic or inhibitor for miR-199b-3p, or U0126 (an inhibitor for the MAPK/ERK/EGR1 axis) were introduced to treat these CMECs. The levels of miR-199b-3p and MAPK/ERK/EGR1 axis-related genes in tissues and cells were detected. The effects miR-199b-3p on the process of CMECs, including cell viability, cell cycle and cell apoptosis were evaluated. miR-199b-3p expressed poorly in the brain tissues after MCAO-R, along with activated MAPK/ERK/EGR1 axis and increased CMECs apoptosis. CMECs transfected with miR-199b-3p mimics and U0126 manifested with increased cell viability, more cells arrested at the S stage, and inhibited apoptosis of CMECs. In conclusion, these key results demonstrated up-regulated miR-199b-3p could protect mice against ischemic stroke by inhibiting the apoptosis of CMECs through blockade of MAPK/ERK/EGR1 axis.

The standardization of the report for urine cell counting-A converting factor for Sysmex UF-1000i.

BACKGROUND: Multicenter laboratory may apply both automated flow cytometer and microscopy for urinalysis. Automated flow cytometer such as Sysmex UF-1000i evaluates particles with native urine without centrifugation and reports as "counts per µL." Microscopic examination recommended as the reference method for urine sediment analysis reports results as "counts per HPF (or µL)." Moreover, some results from flow cytometer are needed to be checked visually under microscopy. Therefore, it is worth to establish the consistency of the results from these two methods. METHODS: Urine specimens from 412 patients were examined with Sysmex UF-1000i and manual microscopy using FAST-READ disposable counting chambers. White blood cell (WBC) and red blood cell (RBC) counting results from UF-1000i after transferred with the converting factor (0.297) we estimated were compared with that from microscopic examination. Method comparison was performed using Passing-Bablok analysis. RESULTS: After transferred with the converting factor (0.297), cell counting results from UF-1000i showed a good correlation with that derived by the reference method (R2 was 0.868 for RBCs (P < 0.001), 0.882 for WBCs (P < 0.001)). Passing-Bablok analysis showed no systematic difference (intercept estimate, -1 [95%CI, -7 to 3] and slightly proportional (slope estimate, 1.2 [95%CI, 1.0 to 1.7]) bias between concentrations of cells measured by manual microscopy and Sysmex UF-1000i using the converting factor. CONCLUSION: The converting factor (0.297) helps to transfer "counts per µL (non-centrifugal urine)" to "counts per µL (equal to centrifugal urine)," and to keep the urine particle analysis results of Sysmex UF-1000i consistent with the results from the reference method.

An Improved Specimen Handling Procedure for Pathogen Detection of the Cerebrospinal Fluid by Microscope.

BACKGROUND: In order to increase the detection rate of pathogenic microorganisms in CSF, an improved specimen handling procedure (ISHP) was created. METHODS: This study enrolled encephalitis and control groups, both groups were handled with traditional specimen handling procedure (TSHP) and ISHP. Glutaraldehyde was added to the ISHP. Observed items included: total protein, glucose, chloride, adenosine deaminase, lactate dehydrogenase, sediment, cell and pathogen, Pandy's test. RESULTS: Sediment test of CSF: There was 1 specimen in 10 control specimens tested by TSHP in which Pandy's test was positive; there were 2 specimens tested by ISHP which could see sediment by eye. There was no statistical difference between those two methods (p = 1.000, Table 1). Ten specimens in 23 of the encephalitis group processed by TSHP were positive with Pandy's test; 23 specimens processed by ISHP could all see sediments by eye (Figure 1). There was a statistical difference between the two methods (p = 0.000, Table 1). Pathogen test of CSF: no pathogen was found in the control group processed by TSHP and ISHP. No pathogen was found in the encephalitis group specimens processed by TSHP. Pathogen tests were positive in 7 encephalitis specimens processed by ISHP (p = 0.009, Table 1), which were confirmed as Rickettsia spp. by Gimenze stain (Figure 1B), IFA (Figure 2). CONCLUSIONS: The results revealed that ISHP contributes to the separation of cells, pathogens (such as Rickettsia), and proteins.

[Screening of meaningful endophytic fungi in Dendrobium officinale based on polysaccharides and flavonoids].

To reveal the relationship between endophytic fungi and the functional components, saccharides and flavonoids in the mycelia or fermented liquor of 21 endophytic fungi in D.officinale were detected using HPLC and UV spectrophotometer.The results showed that the ethyl acetate extracts from 21 fungal strains all contain flavonoids.According to the chromatographic retention time of HPLC and UV spectra characteristics of flavonoids, strain DO49 was found produce naringenin, strains DO23, DO81 and DO83 were found produce rutin.The water-soluble extracts from 21 strains all had polysaccharides.However, there was difference in the composition of monosaccharides derived from polysaccharides among different strains.According to the composition of monosaccharides and the peak area ratio of mannose and glucose, the fungal strains including DO23, DO26, DO81, DO54, DO55, DO83 product polysaccharides associated with D.officinale were selected.In conclusion, based on the saccharides and flavonoids, the excellent endophytic fungal strains DO23, DO81 and DO83 were selected, which could produce the same flavonoids and similar polysaccharides in D.officinale.

Differential expression of plasma miR-146a in sepsis patients compared with non-sepsis-SIRS patients.

Sepsis is a subtype of systemic inflammatory response syndrome (SIRS), which is characterized by infection. Circulating microRNAs (miRNAs), including miR-150, miR-146a and miR-223, are potential biomarkers of sepsis. In this study, we demonstrated that measuring the relative expression of miR-146a/U6 in plasma, using the 2-ΔΔCt method, provides a method for differentiating between sepsis and non-sepsis-SIRS. We observed a significant increase in miR-146a expression in the initial cohort of 6 non-sepsis-SIRS patients compared to the 4 sepsis patients (P=0.01) and in the second cohort of 8 non-sepsis-SIRS patients compared to the 10 sepsis patients (P=0.027). Additionally, we identified that sodium citrate and ethylenediaminetetraacetic acid (EDTA) K2 may be used as anticoagulant reagents. Generation of a standard curve is not necessary in these diagnostic tests, unless the standard of normalization is carefully selected. Thus we provide more detailed guidance for the clinical use of circulating miRNA biomarkers.

The clinical significance comparison of a latex agglutination based syphilis screening test at low antibody titer.

The rapid increase of syphilis underscores a tremendous need to carefully evaluate many new serological tests for syphilis and choose efficient and economical strategies for syphilis screening, especially in the case of primary infection with low antibody titer. Between 2011 and 2012, 73 patients' sera samples were included in this retrospective study. They were either TRUST or TPPA reactive, either LA (latex agglutination) based auto3 TP or CLIA (chemiluminescence assay) based Architect Syphilis TP assay reactive. The contradictory weak response samples were further examined by FTA-Abs method. TPPA could not give reactive results in samples with antibody concentration less than 10 mIU. Auto3 TP reagent shows good linearity at low antibody titers and was more sensitive than TPPA, while the former does not show significant superiority compared to the Architect Syphilis TP assay at low antibody titer, except that it is suitable for adaptation on diverse automated chemistry analyzers.

[Pharmacokinetics of paroxetine tablet in healthy Chinese volunteers].

OBJECTIVE: To study the pharmacokinetics of paroxetine tablet in Chinese healthy volunteers. METHODS: Twenty healthy subjects received a single oral dose of 40 mg paroxetine tablet. The plasma concentrations of paroxetine were determined using high-performance liquid chromatography (HPLC) and the measurements were analyzed with 3P97 program. RESULTS: The plasma concentration curve of paroxetine following a single oral dose administration conformed to the two-compartment open model. The main pharmacokinetics parameters of paroxetine were: C(max)64.74-/+18.43 ng/ml, T(max)5.64-/+1.84 h, t(1/2) 20.03-/+5.33 h, AUC(0-120) 976.47-/+309.49 ng.h/ml, and AUC(0-inf) 1086.75-/+376.54 ng.h/ml. CONCLUSION: The pharmacokinetics of paroxetine in human body conforms to the two-compartment open model.