The Experts below are selected from a list of 90 Experts worldwide ranked by ideXlab platform
Miroslaw łuczak - One of the best experts on this subject based on the ideXlab platform.
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modification and optimization of pcr methods for detection of mie region from Human Herpesvirus 5
Medycyna doświadczalna i mikrobiologia, 2008Co-Authors: łukasz Chabros, Maciej Przybylski, Tomasz Dzieciatkowski, Miroslaw łuczakAbstract:: Human Herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-Herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.
Keith R. Jerome - One of the best experts on this subject based on the ideXlab platform.
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Tolerance of Droplet-Digital PCR vs Real-Time Quantitative PCR to Inhibitory Substances
Clinical Chemistry, 2013Co-Authors: Tanis C. Dingle, Ruth Hall Sedlak, Linda S. Cook, Keith R. JeromeAbstract:To the Editor: Real-time quantitative PCR (qPCR)1 is a rapid and sensitive method that forms the foundation for many clinical diagnostic tests. Droplet digital PCR (ddPCR) shares these qualities with qPCR, but owing to reaction partitioning, ddPCR is proposed to exhibit increased tolerance to interfering substances, making it an attractive alternative to qPCR for diagnostic applications (1, 2). The data to support this phenomenon and its mechanism, however, are currently lacking in the literature (3). Herein, we describe a series of experiments to compare the inhibition tolerance of laboratory-developed CMV qPCR and ddPCR (Bio-Rad Laboratories, QX-100) assays by introducing a panel of clinically relevant inhibitors (SDS, EDTA, and heparin) directly into the PCR reactions (4). Differences in the resulting inhibition curves and the half-maximal inhibitory concentrations (IC50) were then assessed. The laboratory-developed CMV qPCR is a double primer/probe Taqman assay that amplifies and detects the genes UL123 (IE)2 (enhances activation by IE2; interacts with basal transcriptional machinery and cellular transcription factor; disrupts ND10; involved in gene regulation [Human Herpesvirus 5]) and UL55 (gB) (type 1 membrane protein; possible membrane fusogen; binds cell surface heparan sulphate; involved in cell entry; involved in cell-to-cell spread [Human Herpesvirus 5]) with primers and probes previously described (5). The ddPCR assay uses the same primers and probes, with the dyes HEX replacing 6-FAM on the gB probe and BHQ-1 replacing TAMRA on both probes (Sigma-Aldrich). SDS, EDTA, and …
I F S F Boin - One of the best experts on this subject based on the ideXlab platform.
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co infection and clinical impact of Human Herpesvirus 5 and 6 in liver transplantation
Transplantation proceedings, 2012Co-Authors: A M Sampaio, A C Guardia, Arlete Milan, A N Sasaki, Paula Durante Andrade, Sandra Helena Alves Bonon, R S B Stucchi, S Botelho C Costa, I F S F BoinAbstract:BACKGROUND: Human Herpesvirus (HHV) 5 and 6 remain latent after primary infection and can be reactivated after immunosuppression for organ transplantation. An association between HHV-5 and HHV-6 has been reported in liver transplant patients. The coinfection is associated with clinical manifestations and graft dysfunction. OBJECTIVE: The aim of this study was to monitor Herpesviruses in liver transplant recipients to better understand issues involving coinfection with HHV-5/6 and correlations with acute cellular rejection episodes and bacterial infections. METHODS: Forty-five adult liver transplant patients of median age 47 years (range, 18-66), gave blood samples and liver biopsies in the first 6 months after their surgeries. Viremia was detected with the use of nested PCR and antigenemia; the Banff classification was used to detect allograft rejection. RESULTS: IgG positive for HHV-5 was observed in 94% of subjects whose main indication (67%) for transplantation was hepatitis C. Twenty-three (51.1%) displayed cytomeg virus (CMV) infections and 12 (26.7%) HHV-6 infection. There were 6 patients (13.3%) with HHV-5/6 coinfections. Eighteen of the 23 patients had CMV disease, showing a strong correlation between a positive test and CMV disease; 6 displayed an acute cellular rejection episode in the same period (χ(2) = 6.62; P < .03). Four out of 6 patients who displayed coinfections (HHV-5/6) had concomitant bacterial infections; 3/6 experienced graft rejection episodes. During follow-up, 1 patient had HHV-6 infection diagnosed as encephalitis followed by fever on the 24th day after surgery. The median 32 days for HHV-6 detection by nested PCR positivity was shorter than 38 days for HHV-5. CONCLUSIONS: HHV-5/6-infected patients displayed more allograft rejection episodes, coinfections, and concomitant bacterial infections, besides an higher risk for CMV disease.
Bahram Bodaghi - One of the best experts on this subject based on the ideXlab platform.
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Herpes Simplex Virus
Intraocular Inflammation, 2020Co-Authors: Karina Julian, Phuc Lehoang, Bahram BodaghiAbstract:Herpesviridae family comprises more than 100 different members, all of them enveloped DNA viruses with high molecular weight. Among the eight distinct members of this family that infect the Human host (herpes simplex virus types 1 and 2, HSV-1 and HSV-2; varicella-zoster virus, VZV or Human Herpesvirus 3; cytomegalovirus, CMV or Human Herpesvirus 4; Epstein-Barr virus, EBV or Human Herpesvirus 5; Human Herpesvirus 6 (variants A and B); Human herpes virus 7 and Human Herpesvirus 8, HHV-8 or Kaposi’s sarcoma Herpesvirus), herpes simplex are by far the most common ocular pathogens and are responsible for at least 10% of anterior uveitis, regardless of the host’s immune system status.
łukasz Chabros - One of the best experts on this subject based on the ideXlab platform.
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modification and optimization of pcr methods for detection of mie region from Human Herpesvirus 5
Medycyna doświadczalna i mikrobiologia, 2008Co-Authors: łukasz Chabros, Maciej Przybylski, Tomasz Dzieciatkowski, Miroslaw łuczakAbstract:: Human Herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-Herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.