Load In Plasma Samples Research Articles

High silent prevalence of human herpesvirus 1 (HSV-1) infection affecting the indigenous reservation of the municipality of Dourados, Central-West Brazil

BackgroundThe indigenous population located in the central region of Brazil, is the second largest in terms of population size in the country. The Indigenous Reserve of Dourados has risk factors that increase the vulnerability of the indigenous population to infectious diseases, especially Human alphaherpesvirus (HSV-1), a neglected disease with high prevalence in priority populations in developing countries. The virus can also cause many more severe diseases, including widespread neonatal infections, herpetic keratitis, and herpes encephalitis, which can be fatal if left untreated. We estimated the prevalence of anti-HSV-1 antibodies and correlated it with the demographic and behavioral characteristics of the Indigenous population of the Jaguapirú and Bororó villages (Dourados, Mato Grosso do Sul (MS), Brazil).MethodsOur approach was cross-sectional. From March 2017 to November 2018. Using anti-HSV-1 (Gg1) IgM and anti-HSV-1 (gG1) IgG Euroimmun and the detection and quantification of HSV-1 viral load in plasma samples, through real-time PCR. The maps were constructed using QGIS and the statistical analyses using R Studio software.ResultsA total of 1138 individuals (> 18 years old) were enrolled. The prevalence of anti-HSV-1 IgM and IgG were 20% and 97.5%, respectively. The prevalence of anti-HSV-1 antibodies for IgG was higher in both sexes. Anti-HSV-1 IgM antibodies were present in 17.1%, 21.2%, 12.5%, and 22% of the participants with urinary problems, genital wounds, genital warts, and urethral discharge, respectively. Real-time PCR was used for confirmatory testing; HSV-1 DNA was detected in 25.6% (54/211) of anti-HSV1 IgM-positive samples. Viral loads ranged from 5.99E + 02 to 3.36E + 13.ConclusionsThe seroprevalence of HSV-1 IgM and detection of HSV-1 DNA in the Indigenous population confirmed high silent prevalence. Furthermore, the seroprevalence of HSV-1 in the Indigenous population was higher than that reported in the general adult Brazilian population. Various socioeconomic factors, drug use, and health and sexual behaviors could contribute to the facilitation of HSV-1 transmission in the Indigenous population. Our results may help develop culturally appropriate intervention programs that eliminate health access barriers and improve the implementation of public health policies aimed at promoting information regarding the prevention, treatment, and control of HSV-1 infection in Brazilian Indigenous populations.

Analytical and clinical evaluation of the cobas Epstein–Barr virus test at a tertiary care cancer hospital

BackgroundEpstein–Barr Virus (EBV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Cobas EBV test (Roche Molecular, Pleasanton, CA) has recently been FDA-cleared for the monitoring of EBV viral loads in plasma samples of transplant patients. In this study, we compared the viral loads obtained by a laboratory-developed test (EBV LDT) using Altona Analyte specific reagents (ASR) to those obtained on the Cobas EBV test. MethodsThe analytical performance of the assay was established using the EBV verification panel from Exact Diagnostics and the EBV ATCC strain B95-8. The clinical evaluation was performed using 343 plasma samples initially tested on the EBV LDT. ResultsThe analytical sensitivity (<18.8 IU/mL), precision (SD < 0.17 log) and linear range (35.0 IU/mL to 1E + 08 IU/mL) of the Cobas EBV assay established by the manufacturers were confirmed. The strength of the qualitative agreement was substantial between the cobas EBV and the EBV LDT (85.6 %; κ = 0.71) and almost perfect when discordant results were resolved (96.4 %; κ = 0.93). The quantitative agreement was moderate (82.9 %; κ = 0.53) with the viral load obtained on the Cobas EBV test being lower across the linear range of the two tests (mean log difference of 1.0). While the absolute values of the viral loads were markedly different, the overall trends observed in patients with multiple consecutive results were similar between the two tests. ConclusionsThe Cobas EBV test provides an accurate and valid, in vitro diagnostic (IVD) option for monitoring of EBV viral loads in transplant patients and should provide an opportunity for increased standardization and commutability of tests results across laboratories.

The Prevalence of Hepatitis Delta Virus in Patients with Chronic Hepatitis B and Its Association with Risk Factors

Background: Hepatitis B infection is a serious health problem and two billion people worldwide are infected with the virus. The hepatitis delta virus (HDV) is a satellite virus. Hepatitis D virus infection in HBsAg carriers can be present as a simultaneous and acute infection. Objectives: The aim of this study was to evaluate the frequency of HDV in patients with chronic hepatitis B and its association with risk factors. Methods: In this descriptive study, 74 patients with chronic HBV infection were selected from patients that had referred to the Clinical Lab of Blood Transfusion Organization. All patients were positive for HBsAg for more than six months and anti-HBc. All samples were negative for HIV and HCV. An anti-HDV test was performed on HBsAg-positive specimens by the enzyme linked immunosorbent assay (ELISA) method. Also, HBV-real-time polymerase chain reaction (PCR) testing was done to determine the viral load. Results: In this study, 74 HBsAg positive patients with a mean age of 50.22 ± 15.09 years were studied. Five (6.8%) patients had anti-HDV antibodies. Furthermore, 60% of the patients with HDV had risk factors, such as addiction, family history of hepatitis B, and a history of surgery. Maximal viral load in plasma samples of patients with anti-HDV antibodies, 531 IU/mL, was determined. Conclusions: For prevention of HDV transmission, all patients of chronic hepatitis B with low-level viral load should be evaluated for hepatitis D infection. Also, for determining the relationship between HDV infections with its risk factors, another study with a larger sample size should be performed.

One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p = 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values

Performance of a completely automated system for monitoring CMV DNA in plasma

BackgroundCompletely automated systems for monitoring CMV-DNA in plasma samples are now available. ObjectivesEvaluate analytical and clinical performances of the VERIS™/MDx System CMV Assay®. Study designAnalytical performance was assessed using quantified quality controls. Clinical performance was assessed by comparison with the COBAS® Ampliprep™/COBAS® Taqman CMV test using 169 plasma samples that had tested positive with the in-house technique in whole blood. ResultsThe specificity of the VERIS™/MDx System CMV Assay® was 99% [CI 95%: 97.7–100]. Intra-assay reproducibilities were 0.03, 0.04, 0.05 and 0.04 log10IU/ml (means 2.78, 3.70, 4.64 and 5.60 log10IU/ml) for expected values of 2.70, 3.70, 4.70 and 5.70 log10IU/ml. The inter-assay reproducibilities were 0.12 and 0.08 (means 6.30 and 2.85 log10IU/ml) for expected values of 6.28 and 2.80 log10IU/ml. The lower limit of detection was 14.6IU/ml, and the assay was linear from 2.34 to 5.58 log10IU/ml. The results for the positive samples were concordant (r=0.71, p<0.0001; slope of Deming regression 0.79 [CI 95%: 0.56–1.57] and y-intercept 0.79 [CI 95%: 0.63–0.95]). The VERIS™/MDx System CMV Assay® detected 18 more positive samples than did the COBAS® Ampliprep™/COBAS® Taqman CMV test and the mean virus load were higher (0.41 log10IU/ml). Patient monitoring on 68 samples collected from 17 immunosuppressed patients showed similar trends between the two assays. As secondary question, virus loads detected by the VERIS™/MDx System CMV Assay® were compared to those of the in-house procedure on whole blood. The results were similar between the two assays (−0.09 log10IU/ml) as were the patient monitoring trends. ConclusionThe performances of the VERIS™/MDx System CMV Assay® facilitated its routine use in monitoring CMV-DNA loads in plasma samples.

Analysis of the phenotypes and the function of CD56+ T cells during primary HIV-1 infection

Objective To investigate the changes of phenotypes and function of CD56+ T cells during primary HIV-1 infection and their relationship with disease progression. Methods Peripheral blood mononuclear cells (PBMCs) were collected from 53 subjects with primary HIV-1 infection and 31 HIV-1-negative healthy subjects. The percentages of CD56+ T cells and the expression of several phenotypic markers on CD56+ T cells including CD16, CD161, NKB1, NKG2A, NKp46, NKG2D, NKG2C and CD158a were analyzed by flow cytometry. IFN-γ and TNF-α released by CD56+ T cells with and without K562 stimulation and the levels of cytotoxic molecular CD107a were measured. Results The percentages of CD56+ T cells in patients with primary HIV-1 infection were significantly lower than those of healthy subjects (P=0.025). The levels of CD56+ T cells were negatively related to the viral loads in plasma samples (P=0.021, r=-0.316). Compared with healthy subjects, the expression of CD16 (P=0.003), CD161 (P=0.023), NKB1 (P=0.023) and NKp46 (P=0.021) on CD56+ T cells were decreased in patients with primary HIV-1 infection. The levels of NKB1 were positively related to the CD4+ T cell counts (P=0.007, r=0.364), but were negatively related to the viral loads in plasma samples (P=0.030, r=-0.299). Spontaneous secretion of IFN-γ and TNF-α by CD56+ T cells and the expression of CD107a were dramatically inhibited in patients with primary HIV-1 infection as compared with healthy subjects (all P<0.001). Moreover, the killing ability of CD56+ T cells against K562 target cells was weakened in patients with primary HIV-1 infection as the levels of IFN-γ-, TNF-α- and CD107a-producting CD56+ T cells were significantly decreased (P<0.001 for IFN-γ and TNF-α, P=0.016 for CD107a). Conclusion Inhibited expression and altered phenotypes of CD56+ T cells were identified during primary HIV-1 infection. Lower levels of cytokines and cytotoxic molecular were also detected, indicating the dysfunction of CD56+ T cells appeared during early stage of HIV-1 infection and was associated with disease progression. Key words: Human immunodeficiency virus; CD56+ T cells; Phenotype; Cytotoxicity

WHO Collaborating Centre for Acquired Immunodeficiency Syndrome for the Eastern Mediterranean Regional Office, Faculty of Medicine, Kuwait University, Kuwait

In the early 1980s, the World Health Organization (WHO) designated the Virology Unit of the Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait, a collaborating centre for AIDS for the Eastern Mediterranean Regional Office (EMRO), recognizing it to be in compliance with WHO guidelines. In this centre, research integral to the efforts of WHO to combat AIDS is conducted. In addition to annual workshops and symposia, the centre is constantly updating and renewing its facilities and capabilities in keeping with current and latest advances in virology. As an example of the activities of the centre, the HIV-1 RNA viral load in plasma samples of HIV-1 patients is determined by real-time PCR using the AmpliPrep TaqMan HIV-1 test v2.0. HIV-1 drug resistance is determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene, using the TRUGENE HIV-1 Genotyping Assay on the OpenGene® DNA Sequencing System. HIV-1 subtypes are determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene using the genotyping assays described above. A fundamental program of Kuwait's WHO AIDS collaboration centre is the national project on the surveillance of drug resistance in human deficiency virus in Kuwait, which illustrates how the centre and its activities in Kuwait can serve the EMRO region of WHO.

QUANTITATION OF HEPATITIS B VIRUS DNA IN PLASMA USING A SENSITIVE COST-EFFECTIVE “IN-HOUSE” REAL-TIME PCR ASSAY

Background: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an “in-house” real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV. Materials and Methods: The “in-house” real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the “in-house” assay’s capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. Results: The lower limit of detection of this “in-house” HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this “in-house” assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this “in-house” HBV real-time assay correlated well with the commercial artus HBV RG PCR assay (r = 0.95, P < 0.0001). Conclusion: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

A RealTi me HIV-1 viral load assay for automated quantitation of HIV-1 RNA in genetically diverse group M subtypes A–H, group O and group N samples

The Abbott RealTi me HIV-1 assay is an automated test for monitoring HIV-1 viral load in plasma samples. The assay uses reverse transcription polymerase chain reaction (RT-PCR) technology with homogeneous real-time fluorescent detection. Automated sample preparation is performed on the m2000 sp™ instrument where RNA is isolated using magnetic microparticle technology and dispensed to a PCR tray together with the amplification reagents. The PCR tray is then transferred to the Abbott m2000 rt™ instrument for amplification and real-time detection. The assay utilizes two distinct sets of primers and probes for HIV-1 and for internal control (IC). The IC is processed along with each sample to control for sample recovery and inhibition. The HIV-1 primer and probe sequences are targeted to the integrase (IN) region of the polymerase ( pol) gene. Due to the selection of a highly conserved target region and a novel, mismatch tolerant probe design, the assay can quantitate HIV-1 group M subtypes A–H, group O, and group N isolates. The assay provides high reproducibility and a wide dynamic range, allowing quantitation from 40 copies to 10 million copies of HIV-1 RNA per milliliter of plasma. HIV-1 RNA concentrations detected with 95% probability were 25 copies/mL with 1.0 mL of plasma, 39 copies/mL with 0.6 mL of plasma, 65 copies/mL with 0.5 mL of plasma, and 119 copies/mL with 0.2 mL of plasma.

A new Real-Time-RT-PCR for quantitation of human endogenous retroviruses type K (HERV-K) RNA load in plasma samples: Increased HERV-K RNA titers in HIV-1 patients with HAART non-suppressive regimens

Viral components of the human endogenous retroviruses type K (HERV-K) have been largely detected in plasma from HIV-1 infected individuals. A Sybr Green Real-Time RT-PCR approach was optimized for detection and quantitation of HERV-K RNA titers in plasma samples using the iCycler technology. The method detected 1000 HERV-K RNA copies/mL of plasma sample. The Intra- and Inter-assay performance revealed a coefficient of variations that ranged from 0.2 to 2.46%, demonstrating accuracy and reproducibility. We quantified the HERV-K RNA load in 20 HIV-1 patients receiving highly active antiretroviral therapy (HAART). We found increased HERV-K RNA titers in patients with non-suppressive HAART (patients who may develop drug-resistance and/or received suboptimal therapeutic doses), compared to suppressive regimens ( p < 0.001). HERV-K RNA was not detected in HCV-1 positive or seronegative controls. Sequencing of Real-Time RT-PCR products revealed particular HERV-K subtypes activated in the HIV-1 infection. The application of this assay could expand the understanding of the role of HERV-K in the HIV-1 infection and others pathological conditions.

Association of viral load in plasma samples of HIV-infected hemophilia patients with autoantibodies and gp120-containing immune complexes on CD4 + lymphocytes

Objective: We investigated whether the induction of antilymphocyte autoantibodies and immune complexes is associated with the activity of HIV replication. Methods: Viral HIV-1 RNA was measured in the plasma samples of 84 HIV+ hemophilia patients and correlated with the IgM, IgG, IgM/IgG and IgM/IgG/gp120 load of circulating CD4 + lymphocytes, CD4 + and CD8 + cell counts, plasma neopterin levels and in vitro T-cell responses to mitogens and pooled allogeneic stimulator cells. Results: Compared to patients with no immune complexes, on circulating CD4 + lymphocytes, viral load was increased in patients with IgM, IgM/IgG or IgM/IgG/gp120 complexes. Sequential analysis of HIV+ patients showed that peaks of retroviral activity were associated with the subsequent formation of CD4 + lymphocyte-reactive IgM and IgG autoantibodies and gp120-containing immune complexes. Conclusion: The induction of autoantibodies and immune complexes attached to CD4 + lymphocytes is associated with periods of increased viral activity in HIV-infected patients.