Cytokine biomarkers of COVID-19

We used a new strategy to screen cytokines associated with SARS-CoV-2 infection. Cytokines that can classify populations in different states of SARS-CoV-2 infection were first screened in cross-sectional serum samples from 184 subjects by 2 statistical analyses. The resultant cytokines were then analyzed for their interrelationships and fluctuating features in sequential samples from 38 COVID-19 patients. Three cytokines, M-CSF, IL-8 and SCF, which were clustered into 3 different correlation groups and had relatively small fluctuations during SARS-CoV-2 infection, were selected for the construction of a multiclass classification model. This model discriminated healthy individuals and asymptomatic and nonsevere patients with accuracy of 77.4% but was not successful in classifying severe patients. Further searching led to a single cytokine, hepatocyte growth factor (HGF), which classified severe from nonsevere COVID-19 patients with a sensitivity of 84.6% and a specificity of 97.9% under a cutoff value of 1128 pg/ml. The level of this cytokine did not increase in nonsevere patients but was significantly elevated in severe patients. Considering its potent antiinflammatory function, we suggest that HGF might be a new candidate therapy for critical COVID-19. In addition, our new strategy provides not only a rational and effective way to focus on certain cytokine biomarkers for infectious diseases but also a new opportunity to probe the modulation of cytokines in the immune response.

Performance verification of detecting COVID-19 specific antibody by using four chemiluminescence immunoassay systems

BackgroundThe purpose of current study is to evaluate the analytical performance of seven kits for detecting IgM/IgG antibody of corona virus (2019-nCoV) by using four chemiluminescence immunoassay systems. Methods50 patients diagnosed with 2019-nCoV infection and 130 controls without corona virus infection from the General Hospital of Chongqing were enrolled in current retrospective study. Four chemiluminescence immunoassay systems including seven IgM/IgG antibody detection Kits for 2019-nCoV (A_IgM, A_IgG, B_IgM, B_IgG, C_IgM, C_IgG, D_Ab) were employed to detecting antibody concentration. Chi-square test, receiver operating characteristic (ROC) curve and Youdens index were demonstrated to verify the cutoff value of each detection system. ResultsThe repeatability verification results of the A, B, C, and D system are all qualified. D-Ab performances best (92% sensitivity and 99.23% specificity), and B_IgM worse than other systems. Except for the system of A_IgM and C_IgG, the optimal diagnostic thresholds and cutoff value of other kits from recommendations are inconsistent with each other. B_IgM got the worst AUC and C_IgG had the best diagnostic accuracy. More importantly, B_IgG system have the highest false positive rate for testing patients with AIDS, tumor and pregnant. A_IgM system test showed highest false positive rates among elder over 90 years old. ConclusionsSystems for CoVID-2019 IgM/IgG antibody test performance difference. Serum diagnosis kit of D-Ab is the most reliable detecting system for 2019-nCoV antibody, which can be used as an alternative method for nucleic acid testing.

Antibody responses to SARS-CoV-2 in COVID-19 patients: the perspective application of serological tests in clinical practice

BackgroundWe aim to investigate the profile of acute antibody response in COVID-19 patients, and provide proposals for the usage of antibody test in clinical practice. MethodsA multi-center cross-section study (285 patients) and a single-center follow-up study (63 patients) were performed to investigate the feature of acute antibody response to SARS-CoV-2. A cohort of 52 COVID-19 suspects and 64 close contacts were enrolled to evaluate the potentiality of the antibody test. ResultsThe positive rate for IgG reached 100% around 20 days after symptoms onset. The median day of seroconversion for both lgG and IgM was 13 days after symptoms onset. Seroconversion of IgM occurred at the same time, or earlier, or later than that of IgG. IgG levels in 100% patients (19/19) entered a platform within 6 days after seroconversion. The criteria of IgG seroconversion and > 4-fold increase in the IgG titers in sequential samples together diagnosed 82.9% (34/41) of the patients. Antibody test aided to confirm 4 patients with COVID-19 from 52 suspects who failed to be confirmed by RT-PCR and 7 patients from 148 close contacts with negative RT-PCR. ConclusionIgM and IgG should be detected simultaneously at the early phase of infection. The serological diagnosis criterion of seroconversion or the >; 4-fold increase in the IgG titer is suitable for a majority of COVID-19 patients. Serologic test is helpful for the diagnosis of SARS-CoV-2 infection in suspects and close contacts.

A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Corona Virus Disease 2019 (COVID-19)

A respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc.. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.

Comparison and analysis of the detection performance of six new coronavirus nucleic acid detection reagents / 重庆医学

Objective To compare and analyse the detection performance of different 2019-new coronavirus (2019-nCoV) nucleic acid detection kits, in order to provide references for laboratory. Methods Six kinds of domestic reagents (A—F reagent) were selected for parallel detection of a series of samples from one patient in this hospital whose 2019-nCoV nucleic acid result was confirmed weakly positive. The samples were taken at three different times, the RNAs were extracted and amplified, and two parallel tests were performed each time by use of these six kits. The detection performance was compared according to the results of each kit. Results The three parallel test results (ORF1ab and N gene) of C and F reagents were positive, the results of D reagent showed the N gene was not detected, and the results of A, B, E reagents showed the ORF1ab gene was not detected sometimes. The reproducibility of in-batch detections by C reagent was the best, and the CT values of F reagents (N and ORF1ab), E reagents (ORF1ab) and A reagents (ORF1ab) showed changes in trend. Conclusion There are differences in the detection ability of six 2019-nCoV nucleic acid detection reagents for weakly positive samples, and the accuracy, sensitivity and reproducibility of some reagents are not good. There is an urgent need to further optimize and improve their performance in order to better meet the needs of large-scale screening.

Prozone effect on indirect immunofluorescence assay for anti-nuclear antibody testing / 国际检验医学杂志

Objective To explore the influence of prozone effect on anti-nuclear antibodies (ANA) testing by indirect immunofluorescence assay (IIFA).Methods The samples with high titer of ANA (≥1∶1 000) were selected from 880fresh serum samples, and were subsequently diluted in 1∶100, 1∶1 000and 1∶10 000ratio.Prozone effect was defined as fluorescence intensity from 1∶1 000dilution was stronger than that from1∶100dilution.The samples with prozone effect were determined manually or by Sprinter XL and EUROPattern.The samples with prozone effect were further characterized by combinations of fluorescence patterns, fluorescence intensities and autoantibody specificities.Results A total of 880samples were tested.Importantly, 34samples displayed prozone effect (3.86％in total and 29.57％in samples with ANA≥1∶1 000).Interestingly, prozone effect was identified by manual detection as well as by Sprinter XL with similar fluorescence patterns and fluorescence intensities.Notably, EUROPattern can only select the central area for identification.Among all samples with prozone effect, 74.42％samples exhibited fluorescence intensities of≥1∶10 000.Speckled pattern was the most prevalent fluorescence patterns in samples with prozone effect (46.51％).In addition, anti-RNP antibodies (62.79％) were the most popular autoantibodies in samples with prozone effect, followed by anti-dsDNA antibodies (51.16％) and anti-SSA antibodies (51.16％).Conclusion Prozone effect was present in ANA testing, especially in samples with high titers, resulting in underestimating the titers.The study highlighted that special attention should be paid to the prozone effect in clinical practice.

Effect of Mangiferin on Morphology of Relevant Vessels and MCP-1/CCR-2 Pathway in Spontaneously Hypertensive Rats / 中国实验方剂学杂志

Objective:To observe the morphological changes of carotid artery, thoracic aorta and superior mesenteric artery in spontaneously hypertensive rats(SHR), in order to further study the effect of Mangiferin on the expressions of inflammatory factors and monocyte chemoattract protein-1 (MCP-1)/c-chemokine receptor type 2 (CCR-2) pathway in SHR. Method:Forty spontaneously hypertensive rats were randomly divided into model group, benazepril group (10 mg·kg-1·d-1) and low, medium and high-dose mangiferin groups (25, 50, 100 mg·kg-1·d-1). Eight male WKY rats of the same age were selected as normal control group. Systolic blood pressure was observed every two weeks after eight weeks of administration. Morphology of carotid artery, thoracic aorta and superior mesenteric artery was observed by hematoxylin-eosin (HE) staining. Immunohistochemical assay (IHC) and Western blot were used to detect MCP-1 and CCR-2 protein expressions in thoracic aorta. MCP-1 and CCR-2 mRNA expression levels in thoracic aorta were detected by Real-time quantitative fluorescence PCR (Real-time PCR). Result:Compared with the normal group, the inflammatory cells in the model group increased significantly, the systolic blood pressure was significantly higher than that in the WKY group (PPPPConclusion:There are inflammation damages in carotid artery, thoracic aorta and superior mesenteric artery of spontaneously hypertensive rats. Mangiferin has an anti-inflammatory effect by possibly inhibiting the expressions of MCP-1/CCR-2 pathway in SHR vessels.

Investigation on the trueness and precision of routine clinical biochemistry tests in the third-grade class-A hospitals of Chongqing City / 临床检验杂志

Objective@#To investigate the trueness and precision of routine clinical biochemistry tests in the third-grade class-A hospitals of Chongqing city. @*Methods@#The fresh frozen serum samples were assigned the target values with reference methods, and then allocated to the clinical laboratories of the third-grade class-A hospitals in Chongqing city for testing. The trueness and precision were analyzed and evaluated. @*Results@#The pass rates of trueness of creatinine (Cr), total protein (TP), total bilirubin (T-Bil), uric acid (UA) and glucose (Glu) in 33 clinical laboratories were below 50%. The pass rate of trueness of UA (33%) in the closed detection system was lower than that in the opening detection system (79%, P=0.033). In the opening detection system, the pass rate of trueness of Cr in the mode with the same brand of reagents and calibration materials was higher than that with different brands (P=0.014). The precisions of level 1 of T-Bil and Urea in the closed detection system were better than that in the opening detection system (P=0.043 for T-Bil; P=0.031 for Urea). @*Conclusion@#The trueness of clinical biochemistry tests in the third-grade class-A hospitals of Chongqing city needs to be further improved. There is no significant difference in trueness and precision between the opening detection system and the closed detection system, even the performance of some tests in the opening detection system is better than that in the closed detection system.

Human papillomavirus genotype distribution among HPV-positive women in Sichuan province, Southwest China.

In this hospital-based descriptive study, the genotype distribution of human papillomavirus (HPV) among HPV-infected women were investigated in 4,305 gynecological patients in Sichuan province. Females attending gynecology clinics between March 2014 and March 2015 were subjected to HPV screening after giving informed consent. Cervical scrapings were examined by cytopathology and colposcopy-directed biopsies. HPV genotyping was performed on a Luminex 200 system. Seventeen high-risk (HR) genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -66, -68, -73, and -82) and seven low-risk (LR) genotypes (HPV-6, -11, -42, -43, -61, -81, and -83) were detected. Among all HPV-positive women, 34.1% (1,467/4,305) of the cases showed abnormal cytology and biopsy, including high-grade squamous lesions (HSIL), cervical intraepithelial neoplasias of grades 2 and 3 (CIN2/CIN3), and cervical cancer (CC). HPV-16, -52, and -58 were the predominant genotypes, followed by HPV-56, -18, -59, -39, -53, -33, and -81. A total of 3,785 (87.9%) HPV positive women were found to have HR HPV infection, while 859 (20.0%) were found to have LR HPV infection. Among all patients, 79% (3,401/4,305) were infected with a single strain of HPV, 85.5% (2,907/3,401) cases of which were of the HR HPV genotype. In cervical precancerous lesions (CPLs) and CC patients, HR HPV-16, -58, -52, -33, and -18 were the predominant genotypes. Interestingly, 33 CPL patients had a single LR HPV infection with HPV-61, -11, -81, -6, -43, or -42. Furthermore, one CC patient was infected only with LR HPV-11. According to the abundant genotype diversity of HPV in Sichuan, we suggest that a large-scale epidemiological investigation should be launched, not only to understand the distribution of HPV genotype, but also to provide information needed for HPV vaccination programs and to predict the effectiveness of current vaccines in Southwest China.

External Quality Analysis of Quality Indicators on Specimen Acceptability / 现代检验医学杂志

Objective To analyze the status of quality indicators(QI) on specimen acceptability and establish preliminary qual ity specification.Methods Web based External Quality Assessment system was used to collect data of laboratories partici pated in "Medical quality control indicators in clinical laboratory" from 2015 to 2017,including once in 2015 and 2017 and twice in 2016.Rate and sigma scales were used to evaluate incorrect sample type,incorrect sample container,incorrect fill level and anticoagulant sample clotted.The 25th percentile (P25) and 75th percentile (P75) of the distribution of each QI were employed to establish the high,medium and low specification.Results 5 346,7 593,5 950 and 6 874 laboratories sub mitted the survey results respectively.The P50 of biochemistry (except incorrect fill level),immunology and microbiology reach to 6σ.The P50 of clinical laboratory is 4 to 6σ except for incorrect sample container.There is no significant change of the continuous survey results.Based on results in 2017 to establish the quality specification,the P25 and P75 of the four QIs is 0 and 0.084 4 ％,0 and 0.047 6 ％,0 and 0.114 2 ％,0 and 0.078 4 ％,respectively.Conclusion According to the results of the survey,most laboratories had a faire performance in biochemistry,immunology and microbiology,and clinical laboratory needs to be strengthened.Laboratories should strengthen the laboratory information system construction to ensure the actual and reliable data collection,and make a long time monitoring to achieve a better quality.

Clinical distribution and heterogeneity of antimicrobial resistance of Pseudomonas aeruginosa / 中国感染控制杂志

Objective To understand clinical distribution and antimicrobial resistance characteristics of Pseudomonas aeruginosa(P.aeruginosa)isolated from hospitalized patients, so as to provide reference for the empiric use of antimicrobial agents and control of healthcare-associated infection(HAI).Methods Clinical distribution and antimicrobial susceptibility testing results of P.aeruginosaisolated from patients in a hospital between 2012 and 2016 were analyzed retrospectively, statistical analysis were conducted based on different wards, specimen types and age groups.Results A total of 2 432 strains of P.aeruginosa were isolated from2012 to 2016, most of which were isolated from intensive care unit(ICU)(n=727, 29.89％), the main specimen was sputum(n=2 064, 84.87％). Resistance rates of P.aeruginosa to other antimicrobial agents except piperacillin/tazobactam in each year from 2012 to 2016 were significantly different(all P＜0.05).Resistance to piperacillin, ceftazidime, cefepime, imipenem, meropenem, levofloxacin, and ciprofloxacin decreased after peaked in2014;resistance rates to amikacin, gentamicin, and tobramycin were all low, showing decreased trend year by year(all P＜0.05).Except resistance rates to cefepime and tobramycin, resistance rates of P.aeruginosafrom sputum specimen were all higher than other specimens(all P＜0.05).Resistance rates of P.aeruginosaisolated from patients aged≥65 years to most antimicrobial agents were significantly higher than those isolated from patients aged＜65 years(all P＜0.05).Except resistance rates to gentamicin and tobramycin, resistance rates of P.aeruginosaisolated from ICU were higher than those isolated from other departments, which were 7.71％-66.02％.Resistance rate of P.aeruginosaisolated from department of surgery were relatively low, which were 1.69％-11.86％.Conclusion Clinical distribution of antimicrobial resistance of P.aeruginosais obviously heterogeneity, empiric antimicrobial use and formulation of HAI monitoring measures should be based on the data of antimicrobial resistance in different wards, different infection sites, and different age.

Correcting bias of creatinine testing using Jaffe assay by double-levels calibration / 临床检验杂志

Objective To evaluate the bias of creatinine testing by external quality assessment (EQA) and analyze the effects of calibration on the bias of tested values using Jaffe assay.Methods The EQA data from The Clinical Laboratory Centre of Chongqing City were utilized to investigate the frozen pooled-human serum in which the value of creatinine was determined by reference method.The differences between Jaffe assay and creatine oxidase method were analyzed and the bias was evaluated.After the routine measurement systems were calibrated using the frozen pooled-human sera with double levels,the EQA samples were tested.Results The number of the laboratories using Jaffe method for creatinine measurement reduced significantly in Chongqing from 2012 to 2016.The robust average values of Jaffe assay were different from enzymatic assay significantly and the difference of the values were correlated with the concentration of creatinine (r =0.774 6).The results of the investigation using frozen pooled human serum and the trueness verification program of National Center for Clinical Laboratories indicated the positive bias of Jaffe assay was observed in low levels of creatinine with maximum of 10.39％ and 13.31％ and the bias correlated with the concentration of creatinine (r =0.988 9),by contrast the bias was low in enzymatic assay with maximum of 1.04％ and 1.49％ only.After the measurement systems were calibrated by frozen pooled human sera with double-levels,the positive bias of Jaffe assay in the low concentration of creatinine could be corrected.Conclusion There may be a positive bias of Jaffe assay for the samples with low concentration of creatinine,which could be corrected by using the calibrators with double-levels.

Analysis of preoperative,intraoperative,and postoperative bacteriological results and drug sensitivity tests in chronic suppurative otitis media / 重庆医学

Objective To assess the dynamic changes of bacteria culture of patients with chronic suppurative otitis media (CSOM ) preoperative ,intraoperative ,and postoperative the mastoidectomy .Methods Secretions or tissues in 86 CSOM patients were collected for bacterial culture and drug sensitivity tests ,and analyzed the rusults between cholesteatoma group and nonchol-esteatoma group .Results Hospitalized CSOM patients with positive culture rates preoperatively ,intraoperatively and postopera-tively were 75 .6% ,41 .9% and 1 .2% respectively ;Before the mastoidectomy ,positive bacteria ,positive fungus and no pathogenic bacteria were found correspondingly 49 .1% ,25 .0% and 28 .6% of positive bacteria during the surgery .Culture positive rates be-tween the cholesteatoma group and noncholesteatoma group were significantly different intraoperatively (P0 .05) .Different bacteria showed different drug sensitivity results .Conclusion Intraoperative bacteriological results shows the different bacteria and drug sensitivity from the preoperative bacteriological results ;CSOM patients with cholesteatoma are more likely to develop bacteria ;it is necessary to carry out dynamic detection on bacterial culture and drug sensitivity tests before ,during and after the surgery .

Risks of hypoxemia after uvulopalatopharyngoplasty in patients with obstructive sleep apnea syndrome / 中华麻醉学杂志

Objective To observe the risks of hypoxemia after uvulopalatopharyngoplasty (UPPP) in patients with obstructive sleep apnea syndrome (OSAS).Methods Forty-six ASA Ⅱ or Ⅲ male patients with OSAS,aged 30-50 yr,with body mass index 27-33 kg/m2,Mallampati Ⅰ-Ⅳ,underwent UPPP under general anesthesia with propofol and remifentanil.O2 was inhaled for 24 h via a nasal catheter starting from the end of surgery.SpO2 was monitored within 24 h after surgery.Oxygen desaturation index (ODI,hourly average number of desaturation episodes in which the decrease in SpO2 ≥4% and duration ≥ 10 s) and the cumulative time percentage with SpO2 ＜ 90% (CT90) from oximetry were recorded.Results Compared with the baseline value before surgery,ODI and CT90 were significantly decreased at 2 and 2-4 h after extubation and on 1 st night after surgery (11:00 pm-6:00 am) (P ＜ 0.05).ODI and CT90 were significantly lower on 1st night after surgery than at 2 and 2-4 h after extubation (P ＜ 0.05).The rate of ODI abnormalities was 100%,48% and 50% before surgery and at 2 and 2-4 h after extubation,respectively.Compared with the baseline value before surgery,the rate of ODI abnormalities was significantly decreased at 2 and 2-4 h after extubation,while increased on 1 st night after surgery (P ＜ 0.05).There was no significant difference in the rate of ODI abnormalities between that on 1 st night after surgery and that before surgery (P ＞ 0.05).Conclusion Although UPPP can significantly improve airway obstruction in patients with OSAS,hypoxemic episodes still occur after surgery,suggesting that UPPP should not be treated as an ambulatory surgery.

Berberine hydrochloride inhibits formation of Staphylococcus epidermidis biofilm:relative gene expressions / 第三军医大学学报

Objective To investigate the inhibitory effect of Berberine hydrochloride alone or combined with antibiotics on the formation of Staphylococcus epidermidis biofilm and the expressions of relative genes in order to explore a new way to treat the infection induced by the bacteria.Methods Biofilm of Staphylococcus epidermidis was constructed in vitro first,and then Berberine hydrochloride alone or combined with norvancomycin,ciprofloxacin or erythromycin was used to affect the biofilm formation.Then the inhibition was detected by CRA,and the expressions of icaA and agr by the way of fluorescent quantitation RT-PCR.Results The effectiveness of Berberine hydrochloride on the expressions of icaA and agr in Staphylococcus epidermidis was not as better as the other antibiotics.When different-dosed berberine hydrochloride was combined with norvancomycin,ciprofloxacin or erythromycin to affect the biofilm,the inhibition on the expressions of icaA and agr showed a negatively dose-dependent fashion.Conclusion Berberine hydrochloride can inhibit the formation of the biofilm of Staphylococcus epidermidisthrough suppressing the relative genes,though not as good as the antibiotics,such as norvancomycin,ciprofloxacin and erythromycin.It should not be combined with these antibiotics because the effectiveness is antagonism.

Detection of MRCNS and analysis of their drug resistance / 重庆医科大学学报

0.5).The resistance rates of MRCNS to PEN,GEN,SXT,CIP,TET,ALL and ERY,are all above 50%.The resistant rates of MSCNS to other antibiotics are below about 50% except for PEN(80.4%)and ERY(57.1%).MRS are more than 70% and no VAN resistant in CNS.Conclusion:All the 5 methods are suitable for clinical detection of MRCNS ,and latex agglutination is the most convenient and rapid.MRCNS had a serious cross-resistance in aminoglycosides,guinolones,tetracycline,macrolides,sulfanilamide and,so the best choice for therapy is VAN.GEN,SXT,CIP,TET and CLI could be used for MSCNS,except VAN.

Correlation between serum adiponectin and early atherosclerosis in aged patients with type 2 diabetes mellitus / 第三军医大学学报

0.9 mm(n = 26).Twenty-five healthy individuals served as a control group.Clinical data including age,sex,BMI,ABI,HbA1c,fasting glucose and insulin were collected.Insulin resistance index in Homa model was calculated following the equation:Homa IR = FINS ? FPG) /22.5.Serum adiponectin level was measured by ELISA.Results The serum adiponectin level was significantly lower in atherosclerosis group than in non-atherosclerosis and control groups(P