scholarly journals Quantitative Analysis of Human Herpesvirus 8 Viral Load Using a Real-Time PCR Assay

2000 ◽  
Vol 38 (4) ◽  
pp. 1404-1408 ◽  
Author(s):  
Francis Lallemand ◽  
Nathalie Desire ◽  
Willy Rozenbaum ◽  
Jean-Claude Nicolas ◽  
Vincent Marechal
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Symptoms ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Clinical Samples ◽  
Taqman Assay ◽  
Published Data ◽  
Herpesvirus 8 ◽  
Viral Loads

We have developed a quantitative real-time PCR (TaqMan) assay aimed at measuring the cellular human herpesvirus 8 (HHV-8) DNA load in various clinical samples. Standard curves were obtained by serial dilutions of a control plasmid containing both HHV-8 (ORF73 gene) and the cellular target (human albumin gene). The assay appeared to be very sensitive (100% detection rate for at least 10 copies per well) and specific and was easily reproducible (less than 3% intra-assay variability, 5% interassay variability). This method allowed us to quantify precisely the average HHV-8 copy number per cell in various persistently HHV-8-infected cell lines (BBG-1 cells, n= 200; BC-1 cells, n = 59; BCBL-1 cells,n = 70). A retrospective study was also conducted to assess the HHV-8 DNA load in 12 human immunodeficiency virus-infected patients with either Kaposi's sarcoma (KS; seven patients monitored over a 3-month period) or multicentric Castleman's disease (MCD; five patients). The HHV-8 DNA load ranged from 0 to 9,171 copies/106 cells in low-risk KS patients (T0, I0, S0 according to the classification of the AIDS Clinical Trials group). We also measured the viral loads in MCD patients either during symptomatic periods or during remission. The results are in agreement with previously published data, with high viral loads correlating with clinical symptoms (1.3 × 106 copies/106cells) and low viral loads correlating with asymptomatic periods (less than 5,000 copies/106 cells).

scholarly journals Simultaneous quantification of human herpesvirus 8 DNA by real time PCR in different tissues of HIV infected cuban patients with Kaposi's sarcoma

Herpesviridae ◽  
2010 ◽  
Vol 1 (1) ◽  
pp. 3 ◽  
Author(s):  
Vivian Kourí ◽  
Pedro A Martínez ◽  
Orestes Blanco ◽  
Virginia Capó ◽  
María E Rodríguez ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Simultaneous Quantification ◽  
Different Tissues

scholarly journals Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

2002 ◽  
Vol 40 (12) ◽  
pp. 4652-4658 ◽  
Author(s):  
F. Broccolo ◽  
G. Locatelli ◽  
L. Sarmati ◽  
S. Piergiovanni ◽  
F. Veglia ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biological Fluids ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Pcr Assay ◽  
Herpesvirus 8

Development of a LUX real-time PCR for the detection and quantification of human herpesvirus 7

2009 ◽  
Vol 55 (3) ◽  
pp. 319-325 ◽  
Author(s):  
Massimiliano Bergallo ◽  
Cristina Costa ◽  
Maria Elena Terlizzi ◽  
Francesca Sidoti ◽  
Samuela Margio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Primary Infection ◽  
Latent Infection ◽  
Dynamic Range ◽  
Human Herpesvirus ◽  
Clinical Samples ◽  
House Light ◽  
Pcr Assay ◽  
Sensitivity Specificity

Human herpesvirus 7 is a highly seroprevalent β-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

Nonmalignant disease associated with human herpesvirus 8 reactivation in patients who have undergone autologous peripheral blood stem cell transplantation

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2355-2357
Author(s):  
Mario Luppi ◽  
Patrizia Barozzi ◽  
Thomas F. Schulz ◽  
Raffaella Trovato ◽  
Amedea Donelli ◽  
...  
Keyword(s):  
Stem Cell ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Clinical Symptoms ◽  
Human Herpesvirus ◽  
Blood Stem Cell ◽  
Human Herpesvirus 8 ◽  
Pcr Analysis ◽  
Degenerate Primers ◽  
Herpesvirus 8

Fever, cutaneous rash, and hepatitis—for which an infectious cause was suspected—developed in an Italian patient with non-Hodgkin lymphoma after autologous peripheral blood stem cell (PBSC) transplantation. Polymerase chain reaction (PCR) with degenerate primers for the highly conserved DNA polymerase gene of herpesviruses detected herpesvirus sequences 100% identical to human herpesvirus-8 (HHV-8) in serial cell-free serum samples, collected immediately before or concomitant with the occurrence of clinical symptoms; no other common infections were documented. The presence of the HHV-8 genome (clade C) was confirmed by PCR with HHV-8–specific primers for orf 26 and orf-K1. HHV-8 viremia was undetectable either before transplantation or when the patient was clinically asymptomatic. Semiquantitative PCR analysis showed variations of the viral load correlating with the clinical status. Anti–HHV-8 antibodies were detected before and after transplantation by an immunofluorescence assay for lytic antigens. Active HHV-8 infection may be associated with nonmalignant illness after PBSC/bone marrow transplantation.

Nonmalignant disease associated with human herpesvirus 8 reactivation in patients who have undergone autologous peripheral blood stem cell transplantation

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2355-2357 ◽  
Author(s):  
Mario Luppi ◽  
Patrizia Barozzi ◽  
Thomas F. Schulz ◽  
Raffaella Trovato ◽  
Amedea Donelli ◽  
...  
Keyword(s):  
Stem Cell ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Clinical Symptoms ◽  
Human Herpesvirus ◽  
Blood Stem Cell ◽  
Human Herpesvirus 8 ◽  
Pcr Analysis ◽  
Degenerate Primers ◽  
Herpesvirus 8

Abstract Fever, cutaneous rash, and hepatitis—for which an infectious cause was suspected—developed in an Italian patient with non-Hodgkin lymphoma after autologous peripheral blood stem cell (PBSC) transplantation. Polymerase chain reaction (PCR) with degenerate primers for the highly conserved DNA polymerase gene of herpesviruses detected herpesvirus sequences 100% identical to human herpesvirus-8 (HHV-8) in serial cell-free serum samples, collected immediately before or concomitant with the occurrence of clinical symptoms; no other common infections were documented. The presence of the HHV-8 genome (clade C) was confirmed by PCR with HHV-8–specific primers for orf 26 and orf-K1. HHV-8 viremia was undetectable either before transplantation or when the patient was clinically asymptomatic. Semiquantitative PCR analysis showed variations of the viral load correlating with the clinical status. Anti–HHV-8 antibodies were detected before and after transplantation by an immunofluorescence assay for lytic antigens. Active HHV-8 infection may be associated with nonmalignant illness after PBSC/bone marrow transplantation.

scholarly journals Development and evaluation of a multiplex loop-mediated isothermal amplification (LAMP) assay for differentiation of Mycobacterium tuberculosis and non-tuberculosis mycobacterium in clinical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244753
Author(s):  
Jeeyong Kim ◽  
Borae G. Park ◽  
Da Hye Lim ◽  
Woong Sik Jang ◽  
Jeonghun Nam ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Positive Reaction ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Published Data ◽  
Analytical Sensitivity ◽  
Loop Mediated Isothermal Amplification

Introduction The rapid and accurate diagnosis of tuberculosis (TB) is important to reduce morbidity and mortality rates and risk of transmission. Therefore, molecular detection methods such as a real-time PCR–based assay for Mycobacterium tuberculosis (MTB) have been commonly used for diagnosis of TB. Loop-mediated isothermal amplification (LAMP) assay was believed to be a simple, quick, and cost-effective isothermal nucleic acid amplification diagnostic test for infectious diseases. In this study, we designed an in-house multiplex LAMP assay for the differential detection of MTB and non-tuberculosis mycobacterium (NTM), and evaluated the assay using clinical samples. Material and methods For the multiplex LAMP assay, two sets of specific primers were designed: the first one was specific for IS6110 genes of MTB, and the second one was universal for rpoB genes of mycobacterium species including NTM. MTB was confirmed with a positive reaction with both primer sets, and NTM was identified with a positive reaction by only the second primer set without a MTB-specific reaction. Total 333 clinical samples were analyzed to evaluate the multiplex LAMP assay. Clinical samples were composed of 195 positive samples (72 MTB and 123NTM) and 138 negative samples. All samples were confirmed positivity or negativity by real-time PCR for MTB and NTM. Analytical sensitivity and specificity were evaluated for the multiplex LAMP assay in comparison with acid fast bacilli staining and the culture method. Results Of 123 NTM samples, 121 were identified as NTM and 72/72 MTB were identified as MTB by the multiplex LAMP assay. False negative reactions were seen only in two NTM positive samples with co-infection of Candida spp. All 138 negative samples were identified as negative for MTB and NTM. Analytical sensitivity of the multiplex LAMP assay was 100% (72/72) for MTB, and 98.4% (121/123) for NTM. And the specificity of assay was 100% (138/138) for all. Conclusions Our newly designed multiplex LAMP assay for MTB and NTM showed relatively good sensitivity in comparison with previously published data to detect isolated MTB. This multiplex LAMP assay is expected to become a useful tool for detecting and differentiating MTB from NTM rapidly at an acceptable sensitivity.

scholarly journals Quantitation of Cell-Free and Cell-Associated Kaposi's Sarcoma-Associated Herpesvirus DNA by Real-Time PCR

2000 ◽  
Vol 38 (5) ◽  
pp. 1992-1995 ◽  
Author(s):  
Irene E. White ◽  
Thomas B. Campbell
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Kaposi's Sarcoma ◽  
Dynamic Range ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Linear Dynamic Range ◽  
Pcr Assay ◽  
Herpesvirus 8 ◽  
Linear Dynamic

A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 105 copies of KSHV DNA (r 2 > 0.99). The accuracy of DNA purification and quantitation was less than ±0.4 log10copies for samples that contained from 10 to 105 copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0.1 to 10−5, and cell-free KSHV DNA in plasma was quantitated over a range of 100 to 106 copies/ml. Real-time PCR provides a convenient method for quantitation of cell-free and cell-associated KSHV DNA in laboratory and clinical specimens.

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