Diagnostic accuracy of metagenomic next-generation sequencing in diagnosing infectious diseases: a meta-analysis.

Many common pathogens are difficult or impossible to detect using conventional microbiological tests. However, the rapid and untargeted nature of metagenomic next-generation sequencing (mNGS) appears to be a promising alternative. To perform a systematic review and meta-analysis of evidence regarding the diagnostic accuracy of mNGS in patients with infectious diseases. An electronic literature search of Embase, PubMed and Scopus databases was performed. Quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Summary receiver operating characteristics (sROC) and the area under the curve (AUC) were calculated; A random-effects model was used in cases of heterogeneity. A total of 20 papers were eligible for inclusion and synthesis. The sensitivity and specificity of diagnostic mNGS were 75% and 68%, respectively. The AUC from the SROC was 85%, corresponding to excellent performance. mNGS demonstrated satisfactory diagnostic performance for infections and yielded an overall detection rate superior to conventional methods.

Liver Enzyme Elevation in Coronavirus Disease 2019: A Multicenter, Retrospective, Cross-Sectional Study.

INTRODUCTION: Elevated liver enzyme levels are observed in patients with coronavirus disease 2019 (COVID-19); however, these features have not been characterized. METHODS: Hospitalized patients with COVID-19 in Zhejiang Province, China, from January 17 to February 12, 2020, were enrolled. Liver enzyme level elevation was defined as alanine aminotransferase level >35 U/L for men and 25 U/L for women at admission. Patients with normal alanine aminotransferase levels were included in the control group. Reverse transcription polymerase chain reaction was used to confirm severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and patients symptomatic with SARS-CoV-2 infection were defined as patients with COVID-19. Epidemiological, demographic, clinical, laboratory, treatment, and outcome data were collected and compared. RESULTS: Of 788 patients with COVID-19, 222 (28.2%) patients had elevated liver enzyme levels (median [interquartile range {IQR}] age, 47.0 [35.0-55.0] years; 40.5% women). Being male, overweight, and smoking increased the risk of liver enzyme level elevation. The liver enzyme level elevation group had lesser pharyngalgia and more diarrhea than the control group. The median time from illness onset to admission was 3 days for liver enzyme level elevation groups (IQR, 2-6), whereas the median hospitalization time for 86 (38.7%) discharged patients was 13 days (IQR, 11-16). No differences in disease severity and clinical outcomes were noted between the groups. DISCUSSION: We found that 28.2% of patients with COVID-19 presented with elevated liver enzyme levels on admission, which could partially be related to SARS-CoV-2 infection. Male patients had a higher risk of liver enzyme level elevation. With early medical intervention, liver enzyme level elevation did not worsen the outcomes of patients with COVID-19.

Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms.

OBJECTIVE: The SARS-CoV-2-infected disease (COVID-19) outbreak is a major threat to human beings. Previous studies mainly focused on Wuhan and typical symptoms. We analysed 74 confirmed COVID-19 cases with GI symptoms in the Zhejiang province to determine epidemiological, clinical and virological characteristics. DESIGN: COVID-19 hospital patients were admitted in the Zhejiang province from 17 January 2020 to 8 February 2020. Epidemiological, demographic, clinical, laboratory, management and outcome data of patients with GI symptoms were analysed using multivariate analysis for risk of severe/critical type. Bioinformatics were used to analyse features of SARS-CoV-2 from Zhejiang province. RESULTS: Among enrolled 651 patients, 74 (11.4%) presented with at least one GI symptom (nausea, vomiting or diarrhoea), average age of 46.14 years, 4-day incubation period and 10.8% had pre-existing liver disease. Of patients with COVID-19 with GI symptoms, 17 (22.97%) and 23 (31.08%) had severe/critical types and family clustering, respectively, significantly higher than those without GI symptoms, 47 (8.14%) and 118 (20.45%). Of patients with COVID-19 with GI symptoms, 29 (39.19%), 23 (31.08%), 8 (10.81%) and 16 (21.62%) had significantly higher rates of fever >38.5°C, fatigue, shortness of breath and headache, respectively. Low-dose glucocorticoids and antibiotics were administered to 14.86% and 41.89% of patients, respectively. Sputum production and increased lactate dehydrogenase/glucose levels were risk factors for severe/critical type. Bioinformatics showed sequence mutation of SARS-CoV-2 with m6A methylation and changed binding capacity with ACE2. CONCLUSION: We report COVID-19 cases with GI symptoms with novel features outside Wuhan. Attention to patients with COVID-19 with non-classic symptoms should increase to protect health providers.

Clinical findings in a group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) outside of Wuhan, China: retrospective case series.

OBJECTIVE: To study the clinical characteristics of patients in Zhejiang province, China, infected with the 2019 severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) responsible for coronavirus disease 2019 (covid-2019). DESIGN: Retrospective case series. SETTING: Seven hospitals in Zhejiang province, China. PARTICIPANTS: 62 patients admitted to hospital with laboratory confirmed SARS-Cov-2 infection. Data were collected from 10 January 2020 to 26 January 2020. MAIN OUTCOME MEASURES: Clinical data, collected using a standardised case report form, such as temperature, history of exposure, incubation period. If information was not clear, the working group in Hangzhou contacted the doctor responsible for treating the patient for clarification. RESULTS: Of the 62 patients studied (median age 41 years), only one was admitted to an intensive care unit, and no patients died during the study. According to research, none of the infected patients in Zhejiang province were ever exposed to the Huanan seafood market, the original source of the virus; all studied cases were infected by human to human transmission. The most common symptoms at onset of illness were fever in 48 (77%) patients, cough in 50 (81%), expectoration in 35 (56%), headache in 21 (34%), myalgia or fatigue in 32 (52%), diarrhoea in 3 (8%), and haemoptysis in 2 (3%). Only two patients (3%) developed shortness of breath on admission. The median time from exposure to onset of illness was 4 days (interquartile range 3-5 days), and from onset of symptoms to first hospital admission was 2 (1-4) days. CONCLUSION: As of early February 2020, compared with patients initially infected with SARS-Cov-2 in Wuhan, the symptoms of patients in Zhejiang province are relatively mild.

Effect of BZG-4000, a novel multi-targeted kinase inhibitor with potent anticancer activity, on a hepatocellular carcinoma xenograft model.

The present study was to synthesize a novel multi-targeted kinase inhibitor and evaluated its anticancer effects on a hepatocellular carcinoma xenograft model. In our study, in vivo efficacy was determined in nude mice bearing HuH7 human HCC xenografts. The mice were randomly divided into the following five groups with the use of a randomization chart (n = 8 in each group): high-dose BZG-4000 group, medium-dose BZG-4000 group, low-dose BZG-4000 group, sorafenib group, and model group. Tumor size measurements included the length (L) and width (W) measured with calipers, and tumor volume was calculated as (LW∧2)/2. Tumor tissues slides were hematoxylin and eosin (HE) stained for histopathological examination. Immunohistochemistry detected CD31 expression, and Western blotting measured VEGF protein expression. We found that when BZG-4000 was administered orally to xenograft HuH7 nude mice, tumor growth was inhibited and significant tumor shrinkage was evident. After oral administration of BZG-4000 at 40 mg/kg/day, the tumor weight and volume were significantly lower than tumors of the sorafenib group. BZG-4000 considerably decreased the expression of CD31 and VEGF in tumors compared to tumors treated with positive control drug. It was concluded that BZG-4000 has the potential to inhibit the tumorigenesis of hepatocellular carcinoma in vivo by decreasing the expression of CD31 and VEGF.

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ-coupled two-dimensional LC-MS/MS.

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ-coupled 2D LC-MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25-fold at p < 0.05) and 55 proteins were downregulated (<0.8-fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen-like (CD5L), hyaluronan-binding protein 2 (HABP2), and retinol-binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.

Xiao-Chai-Hu Tang in treating model mice with D-galactosamine-induced liver injury.

This study explored the effects of a classical Chinese medicine formula- Xiao-Chai-Hu Tang(XCHT) on the model mice with D-galactosamine -induced liver injury. Sixty male imprinting control region (ICR) mice were used in the present study, and they were separated randomly into 6 groups: a normal control group (Group A, n=10), a model control (Group B, n=10), a positive control (Group C, n=10), a low dose of XCHT group (Group D, n=10), a medium dose of XCHT group (Group E, n=10), and a high dose of XCHT group (Group F, n=10). ELISA was used to detect the IL-6 and TNF-α levels in the serum. Real-time PCR was performed to assess the expression of FasmRNA, Fas-LmRNA, Bcl-2mRNA of the liver tissues. Western blotting was used to detect the Bax protein expression of the liver tissues. The serum IL-6 and TNF-α levels of Group B were significantly higher than the other groups (P<0.05). The expression of Fas mRNA, Fas-LmRNA, and Bax protein of the liver tissues of Group B were significantly higher than those of the other groups (P<0.05). The expression of Bcl-2 mRNA of the liver tissues of Group B was significantly lower than other groups (P<0.05). Both of XCHT and biphenyl dicarboxylate significantly decreased the serum IL-6 and TNF-α levels and FasmRNA, FasLmRNA, Bax protein expression and increased the Bcl-2 mRNA expression of the liver tissues of model mice (P<0.05). It may be through decreasing the serum IL-6 and TNF-α levels and FasmRNA, FasLmRNA, Bax protein expression and increasing the Bcl-2 mRNA expression of the liver tissues that XCHT significantly relieved the D-galactosamine -induced liver injury.

Discovery of potent, orally active compounds of tyrosine kinase and serine/threonine-protein kinase inhibitor with anti-tumor activity in preclinical assays.

Traditional medicines have become the most productive source of leads for drugs development, particularly as anti-cancer agents. Various screening approaches are being applied. Sorafenib, a multikinase inhibitor, is used to treat primary kidney cancer (advanced renal cell carcinoma) and advanced primary liver cancer. A small library of compounds analogous to sorafenib were designed and screened for the treatment of liver cancer. Multiple members of the family in an assay panel of tyrosine kinase family and serine/threonine-protein kinase family, including VEGFR, Abl, Aurora A, p 38, Lck, Src, PDGFR, Flt3, c-RAF, c-KIT, MEK(MAPKK) were selected to test these compounds. Analysis of the selectivity patterns for these compounds shows specificity for many kinase families. IC50 were measured for the selected compounds. Multiple compounds have very similar kinase inhibition profiles of VEGFR, Flt3, FGFR to that of sorafenib. The IC50 of c-RAF of BB1 is lower than sorafenib. The IC50 of c-RAF of BB3-12 is higher than that of sorafenib. For Flt3, IC50 of BB1-4 is less than sorafenib. The IC50 value of KDR of BB1-10 is less than sorafenib. especially against c-RAF, PDGFR, c-KIT, KDR compared to sorafenib. These compounds are potent Raf1 and Flt4 kinase inhibitors.

[The construction and expression of Rv3872 as a member of virulent protein secret system from Mycobacterium tuberculosis in Bacillus Calmette-Guerin].

AIM: To construct, express and identify a recombinant prokaryotic expression vector pET-3872 carrying Rv3872 of Mycobacterium tuberculosis H37Rv strain, and to construct the recombinant Bacillus Calmette-Guerin (rBCG) strain expressing Rv3872 (PE35)of Mycobacterium tuberculosis H37Rv strain. METHODS: The Rv3872 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pET32a(+). The recombinant plasmid pET-3872 was sequenced and transformed into E.coli BL21(DE3), and was induced with IPTG to express a 35 kD fusion protein, which was confirmed as His-Rv3872 by Western blot. The expression product was purified and the new Zealand rabbits were immunized. Rv3872 was amplified by PCR and cloned into E.coli-Mycobacteria shuttle vector pMV361. The recombinant vector was named as pMV-3872, and then pMV-3872 was transformed the to BCG via electroporation, the recombinant BCG-3872 strain. The PE35 protein expression of BCG-3872 was induced with heat shock reaction. Then BCG-3872 culture supernatant and bacterial precipitation were collected respectively and analyzed by Western blot. RESULTS: A recombinant fused expression vector pET-3872 was constructed and His-3872 protein was confirmed by Western blot. BCG-3872 strain was constructed and Western blot confirmed the presenle of the PE35 protein in the supernatant of BCG-3872 strain culture. CONCLUSION: The prokaryotic expression vector pET-3872 was constructed, and the 35 kD fusion protein His-3872 was expressed and purified successfully, which provides a tool for further functional study of the Rv3872 . The recombinant BCG strain expressing PE35 protein, BCG-3872 strain, was constructed successfully, which facilitates further study on BCG-3872 immune function.

Complications associated with the apnea test in the determination of the brain death.

BACKGROUND: An apnea test is essential in the clinical determination of brain death. This study was conducted to analyse complications associated with the apnea test in the determination of the brain death. METHODS: On 93 adult patients in coma in Zhejiang Province of China from January 2003 to December 2006, 179 apnea tests were performed as a part of the determination of brain death. Potential risk conditions and complications were analysed during apnea tests. RESULTS: During apnea, serious cardiac arrhythmia did not occur in all patients. Complications occurred in 37 of 179 (21%) apnea tests. Hypotension occurred in 30 patients (17%) and it was observed in 8/94 (9%) tests with baseline value of systolic arterial blood pressure not less than 120 mmHg, and 22/85 (26%) less than 120 mmHg (P < 0.05). Severe hypoxaemia occurred in 10 patients (6%) of which 3/138 (2%) tests with baseline value of arterial oxygen pressure not less than 200 mmHg, and 7/41 (17%) less than 200 mmHg (P < 0.05). CONCLUSIONS: This study demonstrated that complications occurred mostly in patients with inadequate baseline systolic arterial blood pressure and preoxygenation. Adequate precautions during the apnea tests may reduce the risk of cardiovascular and oxygenation complication.

Pre-existing cirrhosis is associated with increased mortality of traumatic patients: analysis of cases from a trauma center in east China.

AIM: To determine the impact of cirrhosis on trauma patients and define the factors predicting death. METHODS: The data on patients admitted to the trauma center from January 2000-2005 were studied retrospectively. The clinical variables were recorded and compared to identify the factors differentiating cirrhotic trauma survivors from non survivors. Child's classification criteria were derived from the reviewed charts of cirrhotic trauma patients to evaluate their predictive value in cirrhotic trauma. Trauma registry was also used to generate a trauma control group by matching for age, sex, abbreviated injury score (AIS) over the same period of time. The outcome variables compared were mortality rate, time of ICU and hospital stay. Results were expressed as mean +/- SD. These data were analyzed by SPSS.11.0 statistical software. Univariate analysis was performed to identify significant medical factors for survivor and non survivors subjected to chi-square test. Fisher's exact test and Student's t test were performed to determine the statistical difference between cirrhotic and control groups. P < 0.05 was considered statistically significant. RESULTS: Poor prognosis of traum patients was associated with one or more of the following findings: ascitcs, yperbilirubinemia (more than 2 mg/dL), hypoalbuminemia (less than 3.5 mg/dL), and prolonged prothrombin time (more than 12.5 s). Although Child's classification was used to predict the outcome in cirrhotic patients undergoing portacaval shunt procedures, no significant difference was found in mortality rate as a function of Child's classification. CONCLUSION: Cirrhosis is associated with a higher mortality, a longer time of ICU and hospital stay of trauma patients. It seems that treatment of trauma patients with pre-existing severe liver disease is a challenge to surgeons.

[The application of transcranial Doppler in the evaluation of brain function in deeply comatose patients].

OBJECTIVE: To study the cerebral blood flow changes in irreversible comatose patients and investigate the correlation between the results of transcranial Doppler (TCD) and clinical evaluation of brain function. METHODS: TCD studies were performed in 92 patients who were excluded from the possibility of reversible coma and all of them had Glasgow Coma Scale of 3. Their brain stem reflections all disappeared. Detection of the blood flow in bilateral middle cerebral arteries was carried out through temporal window in all the patients and in 42 of the 92 cases the blood flow of the basilar artery was detected through occipital or peri-occipital window. Bilateral middle cerebral artery were taken as the main arteries for evaluation. The positive standard is the appearance of one of the following characteristic blood flow, i.e. brief systolic forward flow or systolic spikes and diastolic reverse flow, brief systolic forward flow or systolic spikes and no diastolic flow and no detectable flow signals. The examination was repeated after 12 h. RESULTS: 68 cases were positive in the initial examination (73.9%) and 74 cases were positive after 12 h (80.4%). CONCLUSION: TCD can be an effective and objective index in the evaluation of irreversible termination of brain function.

[Polymerase chain reaction-single strand conformation polymorphism analysis combined with nucleic acid hybridization to detect ofloxacin-resistant Mycobacterium tuberculosis mutation selected in vitro].

OBJECTIVE: To explore the feasibility of utilizing polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis combined with Southern blot to detect ofloxacin (OFLX)-resistant Mycobacterium tuberculosis, and figure out the boundary between the OFLX-susceptible and resistant Mycobacterium tuberculosis. METHODS: OFLX-resistant Mycobacterium tuberculosis bacilli were induced in vitro and their minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were determined. The 320 bp segments of gyrA gene were amplified by PCR and their polymorphism was tested by SSCP from H(37)Rv, H(37)Ra and those induced OFLX-resistant strains. To prove the repeatability of PCR-SSCP combined with Southern blot, 36 clinical isolated OFLX-resistant strains were tested. RESULTS: All of the 85 OFLX-resistant mutations involved in this study were identified to have gyrA mutations. The results showed that there was an overlap if OFLX 10 microg/ml was used as a criteria for discriminating susceptible and resistant strains. Compared with the H(37)Ra, the similar single-stranded shift caused by the single-stranded conformation change was viewed in the 36 clinical isolated OFLX-resistant strains by PCR-SSCP combined with Southern hybridization. CONCLUSIONS: PCR-SSCP analysis combined with Southern blot can clearly distinguish OFLX-susceptible from OFLX-resistant strains. The results suggest that as a cut-off point between OFLX-susceptible and low-level OFLX-resistant to Mycobacterium tuberculosis, OFLX concentration of 10 microg/ml is considered to be a better one.