Objective:To explore the application of high-throughput sequencing (HTS) technology in pathogens detection for spinal infection.Methods:From January 2019 to May 2020, a total of 41 patients including 31 males and 10 females with an average age of 59.7±11.9 years (29-75 years) were suspected of spinal infections. There were 37 patients with local pain, 15 with fever (≥38 ℃) and 18 with neurological dysfunction. The infected sites were as follows, 4 cases of cervical spine, 8 cases of thoracic spine and 29 cases of lumbar spine. There were 36 patients met the surgical indications and underwent open debridement, bone grafting, fusion and internal fixation, while the other 5 patients underwent conservative treatment (three received drug therapy and two were transferred to the internal department for chemotherapy). Lesions obtained from open surgery patients were underwent pathology and HTS examination. In 5 cases with conservative treatment, two of them underwent CT guided percutaneous puncture for samples, while one case underwent ultrasound guided percutaneous puncture for pus, one case for venous blood, and one case received lumbar puncture for cerebrospinal fluid. The samples were sent for pathological and HTS examination, while liquid specimens were sent for bacterial culture and HTS. The sensitivity and specificity of HTS results were determined according to pathological examination which was regarded as the "gold standard". Based on HTS results combined with the clinical manifestations, imaging examination and pathological results of the patients, targeted antibiotics or anti-tuberculosis drugs were selected for postoperative drug therapy. Patients with bacterial infection received anti-infection treatment for 3 months after operation. For tuberculosis patients, "tetrad" (isoniazid+rifampicin+pyrazinamide+ethambutanol) anti-tuberculosis treatments were underwent for one year. Inflammation indicators from the blood samples were observed before and after treatment, including white blood cell count (WBC), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). These indicators were used to monitor disease progression and the curative effects. All patients were followed up for at least 3 months after surgery.Results:A total of 41 patients with suspected spinal infection were included in this study. The HTS pathogen detection results were obtained within 48 h. For the initial 5 patients, first-generation sequencing verification was conducted with coincidence rate 100%. Further, no further verification was conducted in the rest patients. Among the 41 cases, a total of 26 cases had positive results with a positive rate of 63.4%(26/41). Among them, thirteen cases were with mycobacterium tuberculosis (31.7%) and 6 cases with staphylococcus (14.6%). Fungi and Brucellosis were diagnosed in 2 cases respectively, accounting for 4.9% respectively. The test were negative in 15 patients (36.6%), including 2 patients with tumor or tumor-like lesions (1 hematologic tumor and 1 eosinophilic granuloma). A total of 38 patients underwent pathological examination, which confirmed 7 cases of suppurative infection, 12 cases of tuberculosis, 2 cases of tumor or tumor-like lesions and the remaining 17 cases of inflammatory lesions. The sensitivity and specificity of HTS were 80%(16/20) and 55.6% (10/18) with positive predictive value (PPV) 66.7% (16/24) and negative predictive value (NPV) 71.4% (10/14). All patients were followed up for 3 months. The inflammation indicators of blood at 3 months were all lower than that at admission. WBC decreased from (7.50±3.26)×10 9/L at admission to (6.22±2.53)×10 9/L at 3 months after treatment without statistically significant difference ( t=1.082, P=0.290). The CRP decreased from (32.2±34.1) mg/L to (4.5±10.5) mg/L, and ESR from (44.2±26.5) mm/1 h to (18.6±12.1) mm/1 h with statistically significant difference ( t=8.963, P<0.001; t=5.421, P<0.001). Conclusion:High-throughput sequencing technology can be used in detection of spinal infection pathogens, due to its relatively high positive rate, satisfied sensitivity and good diagnostic value.

Abstract: Objective To establish a rapid detection assay based on fluorescence recombinase polymerase amplification (RPA) targeting Necator americanus eggs, and to evaluate its efficacy, providing technical support for rapid detection of Necator americanus in fecal samples. Methods The fluorescence RPA primers and probe were designed based on the cox1 gene of Necator americanus and then screened the optimal combination to develop the assay. The genomic DNA of Necator americanus eggs was diluted to 7 concentration gradients including 100 pg/µL, 10 pg/µL, 1 pg/µL, 100 fg/µL, 10 fg/µL, 1 fg/µL, 0.1 fg/µL, to determine the detection limit of the assay. The specificity of the assay was demonstrated by detected genomic DNA from Schistosoma japonicum, Ascaris lumbricoides, Clonorchis sinensis and Fasciola hepatica. A total of 44 fecal samples were collected and DNA extraction was performed, and the modified Kato-Katz method, semi-nest PCR method, and fluorescent RPA method were simultaneously used for detection to evaluate the sensitivity and specificity. Results The established fluorescence RPA assay can specifically amplify a fragment of 194 bp of the Necator americanus cox1 gene within 20 min, with a detection limit of 10 fg/µL. There was no cross-reactivity with Schistosoma japonicum, Ascaris lumbricoides, Clonorchis sinensis, Fasciola hepatica after specificity validation. In 44 fecal samples, 27 positive samples were detected by the fluorescence RPA assay, and 26 positive samples were detected by both the Kato-Katz and the semi-nested PCR. The fluorescence curve of sample number 1 was slightly higher than the negative control in the later stage of the reaction, but did not show a similar trend to the positive control, and was therefore judged to be a suspected negative sample. Compared with the Kato-Katz method and the semi-nest PCR method, The sensitivity of the fluorescent RPA method were 100.00% and the specificity were 94.44%, and the consistency of the detection results was good (Kappa=0.953>0.75). Conclusions The assay based on the fluorescence RPA is an efficient, sensitive and specific technique for detecting Necator americanus and it can be applied for surveillance and early warning of hookworm infection.

Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results　The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions　This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.

The history and current situation of cell membrane ion-channel gating mechanism study were reviewed, with an emphasis on the application and the latest developments of kinetic model in gating mechanism study; the problems in present study and ion-channel gating mechanism kinetics model for future investigations were finally discussed.
Cell Membrane ; physiology ; Humans ; Ion Channel Gating ; Kinetics ; Models, Biological ; Research ; trends

Objective To investigate the expression of the HER-2 in human breast carcinomas by comparing the concordance between IHC staining and FISH, and analyze the relationship between the gene expression of HER-2/neu and clinical characteristics of patients. Methods A prospective clinical trial was performed involving a large multicenter patients' samples. Totally 3 249 breast cancer samples were collected from October 2007 to September 2009 in 73 hospitals across mainland China. HER-2 status was assessed by both IHC and FISH using formalin-fixed paraffin sections of consecutive tumor samples. The relationship between the expression of HER-2/neu gene and clinical parameters was analyzed using statistical methods. Results HER-2/neu was detected by FISH in 42.6% (1342/3149) of cases, whereas IHC analysis found 46. 9% (1477/3149) of cases to be HER-2/neu positive (2 +/3 + ). A higher concordance was observed in 94. 1% (892/948) of the patients with scores of 3 + and 89. 9% (660/734) with scores of 0 + by IHC, but relatively low concordance was also observed in 71. 0% (514/725) of patients with scores of 1 + and 55. 9% (415/742) in scores of 2 + by IHC. HER-2/neu gene amplification was associated with negativity of estrogen receptor and progesterone receptor (r = 0. 45, P < 0.01) , high histological grade ( r = 0. 51,P<0.01), more than 4 positive lymph nodes (r =0. 35, P<0.01), advanced stage (r=0. 33, P< 0. 01) , large tumor size ( > 2 cm, r = 0. 38, P < 0. 01 ) , postmenopause (r = 0. 24, P < 0. 01). No statistically significant relationship was found between HER-2 gene status and the other variables including age(r=0.36, P = 0.068), CA125 (r=0.11, P=0.722) or CA153 (r = 0. 23, P=0.45) protein status, lymph node involvement (r=0. 15, P =0. 18), CEA (r = 0.22, P=0.074) , number stage of tumor (r = 0. 21, P = 0.056 ) and blood vessel invasion (r = 0. 12, P = 0. 133 ). Conclusions The comparison of IHC and FISH demonstrated an excellent correlation of HER-2/neu overexpression and gene amplification. The results availabledemonstrate that FISH-analysis as a gold standard should be performed to guide reasonable clinical treatment.

In this paper, the R-PS integration technique of the digital radiology is discussed. By the integration of the RIS and PACS, all data and information of each system and each medical image equipment in R-PS can be exchanged according to DICOM3.0, and seamless linkage can be realized by module interfaces. R-PS has many advantages such as share, safety, compatibility, practicability and feasibility. Standardization of communication interface, modularization of application and resource share of medical information can be realized by this technique.
Computer Communication Networks ; Hospital Information Systems ; Humans ; Radiographic Image Interpretation, Computer-Assisted ; Radiology ; trends ; Radiology Information Systems ; Software ; Systems Integration ; Teleradiology

Objective:To discuss the ameliorative effect of a novel antiepileptic drug Q808 on neuronal injury in temporal lobe epilepsy(TLE)rats,and to clarify its mechanism of action.Methods:TLE rat model was prepared by intraperitoneal injection of the innovative antiepileptic drug candidate 6-(4-chlorophenoxy)-tetrazolo(5,1-a)phthalazine(Q808).Forty-five successfully modeled rats were randomly divided into model group,low dose of Q808 group,and high dose of Q808 group,and there were 15 rats in each group.The rats in low dose of Q808 group and high dose of Q808 group were gavaged with 20 and 80 mg·kg-1 Q808,respectively,and the rats in model group were gavaged with an equal amount of 0.3％sodium carboxymethyl cellulose.Another 15 healthy SD rats were selected as control group.After 4 weeks of continuous gavage treatment,the morphology of the rats in varioius groups was observed;PONEMAH 6.X experimental animal telemetry platform was used to record the electroencephalogram of the rats in various groups;Golgi staining was used to observe the morphology of dendritic and dendritic spine density of hippocampal CA1 neurons of the rats in various groups;Western blotting method was used to detect the expression levels of synaptic plasticity-specific protein calcium/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)in hippocampus tissue of the rats in various groups.Results:The rats in control group showed normal activity without convulsions or other abnormal manifestations.The rats in model group,low dose of Q808 group,and high dose of Q808 group showed varying degrees of reduced activity,trembling and nodding,loss of balance,muscle rigidity and forelimb convulsions,gradually transforming into whole-body muscle rigidity and standing,followed by falling backwards,and there were no convulsions during the interictal period.Compared with control group,the total durations of epileptic seizures of the rats in model group,low dose of Q808 group,and high dose of Q808 group were significantly prolonged(P＜0.01).Compared with model group,the total durations of epileptic seizures in low dose of Q808 group and high dose of Q808 group were significantly shortened(P＜0.01).The hippocampal CA1 neurons of the rats in control group showed regular distribution of dendrites with dense and orderly dendritic networks.The hippocampal CA1 neurons of the rats in model group showed disordered arrangement of dendrites with massive dendritic entanglement,forming thicker nerve fiber bundles.Compared with model group,the dendritic networks of hippocampal CA1 neurons of the rats in low dose of Q808 group and high dose of Q808 group were partially recovered with relatively regular arrangement.Compared with control group,the dendritic spine density of hippocampal CA1 neurons of the rats in model group was significantly decreased(P＜0.01).Compared with model group,the dendritic spine densities of hippocampal CA1 neurons in low dose of Q808 group and high dose of Q808 group significantly increased(P＜0.01).Compared with control group,the expression levels of CaMKⅡ protein in hippocampus tissue of the rats in model group,low dose of Q808 group,and high dose of Q808 group were significantly decreased(P＜0.01).Compared with model group,the expression levels of CaMKⅡ protein in hippocampus tissue of the rats in low dose of Q808 group and high dose of Q808 group were significantly increased(P＜0.01).Conclusion:The novel antiepileptic drug Q808 has an ameliorating effect on the TLE model rats;its mechanism may be related to Q808's ability to reduce the dendritic lesions in hippocampal CA1 neurons and increase the expression level of synaptic plasticity-related protein CaMKⅡ protein.

For patients with chronic hepatitis B, the indications of antiviral treatment are gradually expanding, and the pursuit of various degrees of cure (partial cure, functional cure and complete cure) is consistently improving, which enhances the urgent clinical need for improving the detection sensitivity of hepatitis B virus (HBV) surface antigen and DNA. Based on the availability of commercial highly sensitive hepatitis B surface antigen and HBV DNA detection reagents, we summarized their applications in the diagnosis of HBV infection, their role on guiding selection of antiviral treatment agents and treatment plans, their prediction efficacy and indication for drug withdrawal, their role on monitoring therapy efficacy and the prediction of disease outcomes. Taken together, highly sensitive hepatitis B virus surface antigen and DNA assays and related detection technology play an important role in the whole management process of chronic HBV infection.

Objective:To understand the epidemiological and clinical characteristics of melioidosis in Haikou City, to rise the people's awareness of melioidosis and to provide basis for prevention and control of the disease.Methods:The clinical data of 254 patients with melioidosis treated in 4 Class A tertiary hospitals in Haikou City from January 2000 to September 2020 were collected, and the epidemiological characteristics, clinical manifestations, infection site, prognosis and drug sensitivity were retrospectively analyzed.Results:Among 254 patients with melioidosis, 226 males (88.98%) and 28 females (11.02%), and the gender ratio was 8.07 ∶ 1.00. Farmers were the main occupation, accounting for 37.80% (96/254). The median age was 53 years old, mainly in 41 - 80 years old, accounting for 83.46% (212/254). Han nationality was the most, accounting for 89.76% (228/254). The onset season was mainly in summer and autumn, and the peak was from August to October (117 cases). Patients were mainly distributed in coastal areas, among which Haikou City (49 cases) was the most, followed by Dongfang City (46 cases), Danzhou City (23 cases) and Wenchang City (21 cases). Totally 196 cases (77.17%) had basic diseases, diabetes was the most common (162 cases). The main symptoms of admission were fever (211 cases), followed by cough (108 cases) and expectoration (88 cases). The infection sites were mainly blood (104 cases, 40.94%), lung (60 cases, 23.62%), liver and spleen (32 cases, 12.60%). Totally 195 patients were treated with sensitive antibiotics, at discharge, 37 cases (18.97%) were cured, 129 cases (66.15%) improved, 18 cases (9.23%) did not heal, 7 cases (3.59%) died and 4 cases (2.05%) were discharged voluntarily. Results of drug sensitivity tests from 2010 to 2020 showed that the sensitivity rates of Burkholderia pseudomallei to imipenem (142 cases), meropenem (16 cases) and ceftazidime (141 cases) were all 100.00%, and the sensitivity rates of doxycycline (25 cases) and compound sulfamethoxazole (142 cases) were 92.00% (23/25) and 99.30% (141/142), respectively. Conclusions:Males, farmers, middle-aged and elderly people and people with diabetes and other basic diseases are the high incidence population of melioidosis in Haikou City. The incidence peak is in summer and autumn. The common clinical manifestations are fever, pulmonary infection, abscess of liver and spleen, etc. In the treatment, Burkholderia pseudomallei is more sensitive to imipenem, meropenem and ceftazidime.