Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.

Liver-protecting effects of omega-3 fish oil lipid emulsion in liver transplantation

AIM:To investigate the liver-protecting effect of parenteral nutrition(PN) support with omega-3 fatty acids in a randomized controlled clinical trial.METHODS:Sixty-six patients with the diagnosis of end-stage liver disease or hepatic cellular carcinoma were admitted to the Affiliated Drum Tower Hospital,Nanjing University,China for orthotopic liver transplantation.The patients were randomly divided into two groups:PN group(n = 33) and polyunsaturated fatty acid(PUFA) group(n = 33).All patients received isocaloric and isonitrogenous PN for seven days after surgery,and in PUFA group omega-3 fish oil lipid emulsion replaced part of the standard lipid emulsion.Liver function was tested on days 2 and 9 after surgery.Pathological examination was performed after reperfusion of the donor liver and on day 9.Clinical outcome was assessed based on the post-transplant investigations,including:(1) post-transplant mechanical ventilation;(2) total hospital stay;(3) infectious morbidities;(4) acute and chronic rejection;and(5) mortality(intensive care unit mortality,hospital mortality,28-d mortality,and survival at a one-year post-transplant surveillance period).RESULTS:On days 2 and 9 after operation,a significant decrease of alanine aminotransferase(299.16 U/L ± 189.17 U/L vs 246.16 U/L ± 175.21 U/L,P = 0.024) and prothrombin time(5.64 s ± 2.06 svs 2.54 s ± 1.15 s,P = 0.035) was seen in PUFA group compared with PN group.The pathological results showed that omega-3 fatty acid supplement improved the injury of hepatic cells.Compared with PN group,there was a significant decrease of post-transplant hospital stay in PUFA group(18.7 d ± 4.0 d vs 20.6 d ± 4.6 d,P = 0.041).Complications of infection occurred in 6 cases of PN group(2 cases of pneumonia,3 cases of intra-abdominal abscess and 1 case of urinary tract infection),and in 3 cases of PUFA group(2 cases of pneumonia and 1 case of intra-abdominal abscess).No acute or chronic rejection and hospital mortality were found in both groups.The one-year mortality in PN group was 9.1%(3/33),one died of pulmonary infection,one died of severe intra-hepatic cholangitis and hepatic dysfunction and the other died of hepatic cell carcinoma recurrence.Only one patient in PUFA group(1/33,3.1%) died of biliary complication and hepatic dysfunction during follow-up.CONCLUSION:Post-transplant parenteral nutritional support combined with omega-3 fatty acids can significantly improve the liver injury,reduce the infectious morbidities,and shorten the post-transplant hospital stay.

Expressions and clinical significance of CD147 and CK19 in hepatocellular carcinoma

Objective: The aim of this study was to investigate the expressions of CD147 and CK19 in hepatocellular car- cinoma (HCC) and their clinical significance. Methods: The expressions of CD147 and CK19 were determined by tissue microarray and immunohistochemistry (IHC) in 272 cases of HCC and 81 cases of adjacent tumorous tissue. Results: The positive expression of CD147 in HCC and adjacent tumorous tissue was 73.53% (200/272) and 13.58% (11/81) with signifi- cant difference (P < 0.05). The positive expression of CK19 in HCC and adjacent tumorous tissue was 14.34% (39/272) and 0 (0/81) with significant difference (P < 0.05). The positive expression of CD147 were closely correlated to the histological grade, clinical stage, tumor-free survival, diameter of tumor and embolus of cancer in aqueduct or portal vein; but not to the patients' sex, age, liver cirrhosis, AFP level, infection of HBV, lymph node metastasis, number of tumor, invasion liver involucrum and the micro-satellites (P > 0.05). The expression of CK19 in HCC were closely correlate to the tumor-free survival, histological grade, diameter of tumor, liver cirrhosis, micro-satellites, lymph node metastasis and clinical stage; but not to patients' sex, age, number of tumor, invasion liver involucrum, AFP level, infection of HBV and embolus of cancer in aqueduct or portal vein (P > 0.05). Among the patients of positive expression of CD147, the median replacing time and overall survival were 13 and 24 months, lower than 48 and 60 months in the patients of negative expression (P < 0.05). Among the patients of positive expression of CK19, the median replacing time and overall survival were 7 and 13 months, lower than 31 and 42 months in the patients of negative expression (P < 0.05). The expression of CD147 had no correlation with the expression of CK19 (r = 0.061, P = 0.317). Conclusion: The positive of CD147 and CK19 closely correlate with the clinical prognosis of HCC, it may indicate poor prognosis of HCC.

Determination of the Time of Degradation Trifloxystrobines Fungicide Used in the Apples

Combating the powdery mildew of apples requires permanent intervention with chemical substances. Such interventions are imposed by the development cycle of this pathogen, which during its entire vegetation produces its vegetative reproduction organs-conidia which in turn cause continuous infections. For this purpose, a whole range of fungicides is applied every year, which in order to provide more successful protection is supplemented by new chemical components. One of the most common fungicides which have been largely applied recently in protection of apple cultivars from this disease caused by the pathogen Podosphaera leucotricha is a fungicide produced under the trademark ZATO WG 50, mainly composed of trifloxystrobin. This fungicide also provided the best results in combating this disease during our three-year-long experiment, when compared to other types of fungicides. Taking into account the biology of this pathogen, this fungicide should be introduced until the end of vegetation, which may raise suspicion of its degradation into the apple fruits. In our study, apart from measuring the intensity scale of infection with this disease, we also conducted analyses of the time needed for trifloxystrobin to degrade into the apple fruits, by accurately determining the time of its decomposition by its application into two different concentrations.

Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-time Quantitative Reverse Transcription-PCR Assays

The etiological agent of severe acute respiratory syndrome(SARS) was identified as a new coronavirus,termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study,we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreenTM dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities,13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase(GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection,but because of its sequence variability in the different viral strains,primers and a probe based on the N gene were suitable substitutions. Meanwhile,we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

Extra-pancreatic complications,especially hemodialysis predict mortality and length of stay,in ICU patients admitted with acute pancreatitis

Background and aims:Patients in the intensive care unit(ICU)with acute pancreatitis(AP)are at risk for extra-pancreatic complications given their severe illness and prolonged length of stay.We sought to determine the rate of extra-pancreatic complications and its effect on length of stay(LOS)and mortality in ICU patients with AP.Methods:We performed a retrospective cohort study of ICU patients admitted to a tertiary-care center with a diagnosis of AP.A total of 287 ICU patients had a discharge diagnosis of AP,of which 163 met inclusion criteria.We calculated incidence rates of extra-pancreatic complications and performed a univariate and multi-variable analysis to determine predictors of LOS and mortality.Results:There were a total of 158 extra-pancreatic complications(0.97 extra-pancreatic complications per patient).Ninetyfive patients had at least one extra-pancreatic complication,whereas 68 patients had no extra-pancreatic complications.Patients with extra-pancreatic complications had a significantly longer LOS(14.7 vs 8.8 days,p<0.01)when controlling for local pancreatic complications.Patients with non-infectious extra-pancreatic complications had a higher rate of mortality(24.0%vs 16.2%,p=0.04).Patients requiring dialysis was an independent predictor for LOS and mortality(incidence risk ratio[IRR]1.73,95%confidence interval[CI]:1.263–2.378 and IRR 1.50,95%CI 1.623–6.843,p<0.01)on multi-variable analysis.Coronary events were also a predictor for mortality(p=0.05).Other extra-pancreatic complications were not significant.Conclusions:Extra-pancreatic complications occur frequently in ICU patients with AP and impact LOS.Patients with noninfectious extra-pancreatic complications have a higher mortality rate.After controlling for local pancreatic complications,patients requiring dialysis remained an independent predictor for LOS and mortality.

Determination of epidemic parameters from early phase fatality data： A case study of the 2009 A（H1N1） pandemic in Europe

This paper demonstrates that the susceptible-infected-removed （SIR） model applied to the early phase of an epidemic can be used to determine epidemic parameters reliably. As a case study, the SIR model is applied to the fatality data of the 2009 fall wave cycle of the A（H1N1） pandemic in 12 European countries. It is observed that the best estimates of the basic reproduction number R0 and the mean duration of the infection period l/r/ lie on a curve in the scatterplots, indicating the existence of a nearly-invariant quantity which corresponds to the duration of the epidemic. Spline interpolation applied to the early phase of the epidemic, an approximately 10-week period, together with a future control point in the stabilization region, is sufficient to estimate model parameters. The SIR model is run over a wide range of parameters and estimates of R0 in the range 1.2- 2.0 match the values in the literature. The duration of the infection period, 1/η is estimated to be in the range 2.0-7.0 days. Longer infection periods are tied to spatial characteristics of the spread of the epidemic.

Stability and multi-parametric Hopf bifurcation analyses of viral infection model with time delay

Ever since HIV was first diagnosed in human, a great number of scientific works have been undertaken to explore the biological mechanisms involved in the infection and progression of the disease. This paper deals with stability and bifurcation analyses of mathematical model that represents the dynamics of HIV infection of thymus. The existence and stability of the equilibria are investigated. The model is described by a system of delay differential equations with logistic growth term, cure rate and discrete type of time delay. Choosing the time delay as a bifurcation parameter, the analysis is mainly focused on the Hopf bifurcation problem to predict the existence of a limit cycle bifurcating from the infected steady state.Further, using center manifold theory and normal form method we derive explicit formulae to determine the stability and direction of the limit cycles. Moreover the mitosis rate r also plays a vital role in the model, so we fix it as second bifurcation parameter in the incidence of viral infection. Our analysis shows that, while both the bifurcation parameters can destabilize the equilibrium E＊ and cause limit cycles. Numerical simulations are performed to investigate the qualitative behaviors of the inherent model.

STOCHASTIC DISCRETE-TIME AGE-OF-INFECTION EPIDEMIC MODELS

We present an epidemic model which can incorporate essential biological detail as well as the intrinsic demographic stochastieity of the epidemic process, yet is very simple, enabling rapid generation of a large number of simulations, A deterministic version of the model is also derived, in the limit of infinitely large populations, and a final-size formula for the deterministic model is proved. A key advantage of the model proposed is that it is possible to write down an explicit likelihood functions for it, which enables a systematic procedure for fitting parameters to real incidence data, using maximum likelihood.

THE DELAYED BARBOUR＇S MODEL FOR SCHISTOSOMIASIS

The transmission of schistosomiasis involves latent periods of infected hosts. In this paper, considering the latent periods of infected human, infected bovines and infected snails, we propose a delayed Barbour＇s model with two definitive hosts and define basic reproductive number. The stability of equilibria for the systems with and without time delays are both investigated. To study the impact of the latent periods on the transmission of schistosomiasis, some sensitivity analysis of the basic reproductive number on the three time delays are carried out. It is shown that the basic reproductive number decreases as the three time delays increase. Furthermore, the impact of the latent periods of infected snails on the system is stronger than that of the latent periods of infected human and infected bovines. Thus, to reduce the prevalence of schistosomiasis infection, prolonging the latent periods of infected snails by some measures could achieve better results than prolonging the latent periods of infected definitive hosts.

Dynamical analysis of a class of hepatitis C virus infection models with application of optimal control

In this paper, we deal with the problem of optimal control of a deterministic model of hepatitis C virus （HCV）. In the first part of our analysis, a mathematical modeling of HCV dynamics which can be controlled by antiretroviral therapy as fixed controls has been presented and analyzed which incorporates two mechanisms： infection by free virions and the direct cell-to-cell transmission. Basic reproduction number is calculated and the existence and stability of equilibria are investigated. In the second part, the optimal control problem representing drug treatment strategies of the model is explored considering control parameters as time-dependent in order to minimize not only the population of infected cells but also the associated costs. At the end of the paper, the impact of combination of the strategies in the control of HCV and their effectiveness are compared by numerical simulation.