Objective Liver inflammatory activity staging is critical to guide the treatment of chronic hepatitis B virus (CHB) infection. Here, we aimed to identify practical clinical biomarkers of moderate inflammatory activity in hepatitis B e-antigen (HBeAg)-negative CHB patients. Methods Treatment-naïve HBeAg-negative CHB patients who underwent liver biopsy at our hospital from 1 January 2013 to 31 December 2016 were enrolled. Markers of inflammatory activity were analyzed using binary logistic regression. The area under the receiver operator characteristic curve (AUROCC) was used to assess diagnostic accuracy. Results A total of 106 HBeAg-negative treatment-naive CHB patients were enrolled. According to their METAVIR inflammatory scores, 30.2% of patients were in stage ≥A2. Total triiodothyronine (TT3) and hepatitis B virus (HBV) DNA levels were predictors of moderate inflammatory activity (A ≥ 2). The AUROCCs of TT3 and HBV DNA levels were 0.651 and 0.797, respectively. The optimal cut-off values for TT3 and HBV DNA were 1.755 nmol/L and 4.61 log10 IU/mL, respectively. Conclusions A sizable proportion of treatment-naive HBeAg-negative CHB patients required antiviral treatment (30.2%) after undergoing liver biopsy. TT3 and HBV DNA helps identify patients with moderate inflammatory activity (A ≥ 2), potentially reducing the need for liver biopsies and helping guide treatment of CHB patients.
Cytomegalovirus (CMV) infection in patients after autologous stem cell transplant (ASCT) has become a major medical concern. However, few studies have reported active CMV infection before the transplantation. In this study, we retrospectively analyzed the incidence, clinical impact, risk factors, and outcome of CMV pp65 antigenemia in 44 consecutive patients who underwent ASCT between January 2005 and June 2011. CMV pp65 antigenemia assay was performed weekly, from 7 days before stem cell infusion until the patient was discharged. All patients were CMV seropositive before ASCT. Pre-transplantation antigenemia was detected in 19 patients (43.2%) and post-transplantation antigenemia in 17 patients (38.6%). Multivariate analysis could not identify any pre-transplantation risk factors for CMV antigenemia. When patients with and without pre-transplantation antigenemia were compared, we found that pre-transplantation active CMV infection had significant effects on post-transplantation platelet and neutrophil recovery, although the time of hospitalization and amount of blood transfusion were similar in both groups. CMV antigenemia was asymptomatic in all cases, and cleared spontaneously in 86.7% of patients who did not receive antiviral treatment. In conclusion, CMV reactivation before ASCT might occur frequently and even an asymptomatic infection could significantly delay engraftment. Therefore, ASCT candidates should be routinely evaluated for active CMV infection.
Key words: Cytomegalovirus, autologous stem cell transplant, CMV pp65 antigenemia, infection
The BK virus is the most common pathogen in renal transplant recipients. Limited information is available regarding JC virus or Simian virus infections in renal transplant recipients. This prospective study sought to investigate the rate of BK virus, JC virus, and Simian virus 40 infections and their influence on allograft function in the early stages after surgery.In total, 50 renal transplant recipients and 20 healthy blood donors were studied. The BK virus, the JC virus, and the Simian virus 40 were detected by nested qualitative polymerase chain reaction assays in urine and plasma. The difference of glomerular filtration rate among BK virus-infected, JC virus-infected, and uninfected patients was compared using the Kruskal-Wallis test.The polyomavirus viruria was detected in 46% of renal transplant recipients (4% of the BK virus and 42% of the JC virus viruria) and 10% of the healthy blood donors (5% for the BK virus and the JC virus viruria). No polyomavirus viremia was detected. No difference of glomerular filtration rate was found among the 3 groups (chi-square = 0.228; P = .892).Polyomavirus infections are not uncommon, and the incidence of JC virus infection is much higher in renal transplant recipients than it is in BK virus. Neither BK virus nor JC virus infections appeared to influence graft function in the early stages after surgery.
Vibrio vulnificus is a common gram-negative bacterium, which might cause morbidity and mortality in patients following consumption of seafood or exposure to seawater in Southeast China. We retrospectively analyzed clinical data of patients with laboratory confirmed V. vulnificus infection. Twenty one patients were divided into a survival group and a non-surviving (or death) group according to their clinical outcome. Clinical data and measurements were statistically analyzed. Four patients (19.05%) died and five patients gave positive cultures from bile fluid, and 16 other patients gave positive culture from blood or blisters. Ten patients (47.62%) had an underlying liver disease and marine-related events were found in sixteen patients (76.2%). Patients with heavy drinking habits might be at increased mortality (p = 0.028). Clinical manifestations of cellulitis (47.6%), septic shock (42.9%) and multiple organ failure (28.6%) were statistically significant when comparing survivors and non-survivors (p = 0.035, p = 0.021 and p = 0.003, respectively). The laboratory results, including hemoglobin < 9.0 g/L (p = 0.012), platelets < 2.0×109 /L, prothrombin time activity (PTA) <20%, decreased serum creatinine and increased urea nitrogen were statistically significant (p = 0.012, p = 0.003, p = 0.028 and p = 0.028, respectively). Patients may be at a higher risk of mortality under situations where they have a history of habitual heavy alcoholic drink consumption (p = 0.028, OR = 22.5, 95%CI 1.5–335.3), accompanied with cellulitis, shock, multiple organ failure, and laboratory examinations that are complicated by decreased platelets, hemoglobin and significantly prolonged prothrombin time (PT).
The aim of the present study was to investigate the effects of paclitaxel (PTX), at a non-cytotoxic concentration, on pulmonary vascular remodeling (PVR) in rats with pulmonary hypertension (PAH), and to explore the mechanisms underlying the PTX-mediated reversal of PVR in PAH. A total of 36 rats were divided into control group (n=12), model group (n=12) receiving a subcutaneous injection of monocrotaline (60 mg/kg) in the back on day 7 following left pneumonectomy and PTX group (n=12) with PTX (2 mg/kg) injection via the caudal vein 3 weeks following establishing the model. The degree of PVR among all groups, as well as the expression levels of Ki67, p27Kip1 and cyclin B1, were compared. The mean pulmonary artery pressure, right ventricular hypertrophy index [right ventricle/(left ventricle + septum) ratio] and the thickness of the pulmonary arterial tunica media in the model group were 58.34±2.01 mmHg, 0.64±0.046 and 65.3±3.3%, respectively, which were significantly higher when compared with 23.30±1.14 mmHg, 0.32±0.028 and 16.2±1.3% in the control group, respectively (P<0.01). The mean pulmonary artery pressure, right ventricular hypertrophy index and thickness of the pulmonary arterial tunica media in the PTX group were 42.35±1.53 mmHg, 0.44±0.029 and 40.5±2.6%, respectively, which were significantly lower when compared with the model group (P<0.01). Compared with the control group, the expression levels of Ki67 and cyclin B1 in the model group were significantly increased (P<0.01), while p27Kip1 expression was significantly reduced (P<0.01). Following PTX intervention, the expression levels of Ki67 and cyclin B1 were significantly reduced when compared with the model group (P<0.01), while p27Kip1 expression was significantly increased (P<0.01). The results of the present study suggest that PTX, administered at a non-cytotoxic concentration, may reduce PAH in rats, and prevent the effects of PVR in PAH. These effects of PTX may be associated with increased expression of p27Kip1 and decreased expression of cyclin B1.
Cytomegalovirus (CMV) infection is a major and often deadly complication of haematopoietic stem cell (HSC) transplantation.1 Successful preemptive CMV therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections.2 The pp65 antigenemia assay has been used for this purpose with considerable success but has disadvantages of being time-consuming and labor-intensive.3 Recently, commercial quantitative polymerase chain reaction (PCR) methods based on TaqMan technique have become available and proven to be useful in the diagnosis of microbial infection as well as the determination of viral load.4 In this study, we developed a fluorescent-based quantitative real-time PCR (RT-FQ PCR) assay using TaqMan chemistry for rapid and quantitative detection of CMV DNA and assessed its clinic value for monitoring the reinfection of CMV in patients after HSC transplantion. METHODS Patients Sixty-eight recipients, 51 men and 17 women with the average age (33.5 ± 12.8) years (range 15—48), who underwent HSC transplantation at our hospital during September 2002 to January 2005, were included in this study. Blood specimens were collected and used for detecting the CMV DNA load in plasma and the CMV pp65 expression in leukocytes, respectively. Eight healthy men with the average age (28.5 ± 13.2) years (range 18 — 45) were distributed to the control group. Immunohistochemical staining A standard two-step immunohistochemical method was used to detect the CMV antigen expression in peripheral blood leukocytes. In brief, leukocytes was separated from EDTA anti-coagulated blood and spread on slides. Anti-CMV-PP65-Ag monoclonal antibody (DAKO, Denmark) and EnVision™ +System, peroxidase (DAB) Kit (DAKO, Denmark) were used. The stained samples were analyzed under optical microscope accompanied with imaging recording system (BH-2, OLYMPUS, Japan). Cells stained yellow or brown were positive while blue were negative. Results were indicated as positive cell number per 5 × 104 leukocytes. Establishment of RT-FQ PCR assay Quantification standard A PUCm-CMV plasmid containing CMV UL123 gene, kindly provided by Dr. SHAO Jun-bin from Shanghai Zhijiang Biotechnology Incorporation, was used as the quantification standard for the fluorescent real-time PCR assay. Plasmid DNA was extracted by S.N.A.P Miniprep Kit and quantified by spectrophotometry (Pharmacia Biotech, USA). The quality of DNA sample was also checked by spectrophotometry and reached OD260/OD280 >1.7. Serial dilutions, starting from 26 ng/ml (1 × 1010 copies/ml) of this plasmid DNA was used to create the external calibration curve by which to quantify the viral load in fluorescent real-time PCR. Sample DNA preparation CMV-DNA was extracted from peripheral blood leukocytes by using virus DNA extraction kit (Qiagen, USA) following the manufacturer's instruction. PCR programme The CMV-specific PCR primers (Bioreseach INC.) were as follows: CMV-F 5′-CCAGTGCCCGCAGTTT TTATT-3′, CMV-R 5′-ACCGGAGAAGAGCCCATGT C-3′, and CMV-P 5′-FAMAACATAACGTGGGATCT CCACGCGAATTAMARA-3′. The TaqMan probe was labeled at the 5′ end with the fluorescent reporter FAM and at the 3′ end with the fluorescent quencher BHQ1. CMV-F and CMV-R 0.6 μmol/L, CMV-P 0.3 μmol/L, 10×Taqman reaction buffer 4 μl, MgCl2 5.0 mmol/L, dNTP 200 nmol/L, Taq DNA polymerase 1.5 U, and template 4 μl were added to 40 μl of PCR mixture. The PCR was performed on an ABI-7000 Thermal Cycler (Applied Biosystem). Template denaturation for 5 minutes at 95°C was followed by 40 cycles of denaturation at 93°C for 10 seconds, annealing at 60°C for 40 seconds. Fluorescence data were obtained during the annealing period with the "single" mode and the channel setting F1 (530 nm) within the LightCycler software package. Statistical analysis The SPSS10.0 analysis statistics software was used. Comparison of categorical variable was performed by Chi-square test. P<0.05 was considered statistically significant. RESULTS The dynamic range and detection limit of CMV DNA RT-FQ PCR assay The quantitative real-time PCR assay for PUCm-CMV, namely CMV UL123 gene was linear over a 7 log dynamic range, from 108 copies/ml to the lower detection limit of 102 copies/ml (R=-0.999, Fig). The detection limit was determined by using three CMV standard-curve DNA dilutions with 10 copies, 50 copies, and 100 copies per PCR reaction, respectively in 10 distinct experiments. In six often attempted experiments, 50 copies per PCR reaction were detected, and 10 molecules were detected in two of ten replicate experiments. In comparison, a CMV template quantity of 100 copies or above was always positive by PCR in those ten attempted replicates. Thus the lower limit of quantitative assay was determined as 100 copies/ml.Fig.: CMV RT-fQ PCR. A: raw amplification curves from one experiment. The curves represent input plasmid copy numbers of (left to right) 108,107, 106, 105, 104, 103, 102 molecules per PCR reaction. B: standard curve, R=—0.999.CMV DNA load Thirty-seven of 68 (54.4%) recipients showed CMV DNA positive with the virus load between 0 to 7.19 × 106 copies/ml. The mean first checkout time was (29.3 ± 3.6) days. The results for control group were all negative. CMV antigenemia Thirty-five of 68 (51.5%) recipients showed antigenemia with the positive cell number 1 —28 per 5×104 white blood cells. The mean first checkout time was (32.1 ±2.5) days. In contrast, none of the healthy donor was detected CMV-Ag expression. Comparison of pp65 antigenemia and CMV DNA The level of CMV DNA detected by real-time PCR and the number of CMV antigen-positive cells by the antigenemia assay correlated well (R=0.811). Results comparison between CMV DNA and pp65 antigenemia revealed that 31 patients were positively (45.6%) detected by both assays while 27 were negative (39.7%) in either assay. The coincidence was 85.3% (χ2=0.12, P>0.05), which was considered no statistical significance. (Table)Table: Comparison between CMV DNA and pp65 antigenemia diagnosis in patients after haematopoietic stem cell transplantation (n =68)DISCUSSION CMV can latently infect healthy adults without clinical symptom. In transplant including HSC transplant recipients, partially due to the impaired immune situation of the patient and the administration of the immunosuppressive drugs; the latent virus is high likely reactivated and responsible for server and often life threatening disease. Potentially the reactivation of CMV is the third main cause of mortality in those patients during the first three months after operation.5 Preemptive therapy, which is commonly used to prevent the development of CMV infection after transplantation, is dependent upon the availability of appropriate diagnostic tests for early stages of CMV infections.6 In general, the replicative cycle of CMV could be divided into three stages: the immediate-early phase (IE), the early phase (E), and the late phase (L). The pp65 protein was synthesized at L phase. Its antigenemia assay has been used for early detection of CMV active infection with considerable success.7 However, this assay is labor-intensive, easy false positive and time-consuming. The whole procedure needs three days. Besides, it requires samples to be processed within a few hours. In addition, its reading is subjective and therefore requires skilled interpretation.6 The recently developed RT-FQ PCR assay is a sensitive method for detection of microbial infection.8 In this study, we introduced self-designed fluorescent-labeled TaqMan probes into the PCR procedure. The fluorescent quencher located in the 3' end could efficiently inhibit the activity of the fluorescent reporter located in the 5' end without interfere the integrity of the probes. During the course of prolongation, the fluorescence of Taq polymerase group R was detected and recorded. The density of fluorescence, which is associated with the amount of PCR produces, was automatically recorded and qualitatively analyzed by the LightCycler software package. It came out with the shortened time and labor, and reduced artificial mistakes as well.9 Our RT-FQ CMV PRC assay showed high sensitivity with the dynamic range of detection being between 108 copies/ml and 102 copies/ml. Since the virus load detecting is taken place in an enclosed system within a single step, the gel electrophoresis and the potent cross-species contamination of regular PCR might be avoided, by which promised the accuracy of the results.10,11 IE gene of CMV consists of CMVUL123 gene. The copy number of CMV UL123 gene was detected in 68 patients after HSC transplantation by RT-FQ PCR assay. The detection limit was 102 copies/ml. The positive rate of this assay vs pp65 antigen by immunohistochemistry was 54.4% and 51.5% respectively. The positive rate by two assays was 45.6% and the negative rate was 39.7%. There was no significant difference. The coincidence of two assays was 85.3%. These results indicate that our RT-FQ PCR assay is reliable to monitor CMV copies after operation. The newly designed RT-FQ PCR assay had a great capacity and it was accurate, rapid and labor-saving relative to immunohisto-chemistry for CMV detection. The quantified CMV DNA copies indicated the virus load. So the TaqMan real-time PCR assay is able to detect CMV reactivation in HSC transplant recipients as well as pp65 antigenemia assay. However, results from ten samples by two assays were not consistent. Since UL123 gene copy and pp65 protein expression were in different replicative stage, it may lead to DNA positive but pp65 protein negative. Our results also demonstrated that the occurrence of CMV DNA was earlier than that of antigenemia. On the other hand, DNA negative but pp65 positive may be caused by the false positive of immunohist-ochemistry or blockage of CMV DNA copies by antivirus therapy. The standard threshold of UL 123 gene of CMV active infection is contradictory. At present, each laboratory uses its own standard threshold in RT-FQ PCR. We established real-time quantitative PCR assay preliminarily in this study and our further work will focus on the establish of the Chinese standard threshold of UL 123 gene of CMV active infection after bone marrow transplant. It will be significance for clinical timely prevention and treatment of CMV infection.
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