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Epstein-Barr virus load in the peripheral blood of patients with rheumatoid arthritis: accurate quantification using real-time polymerase chain reaction.

OBJECTIVE: To determine whether patients with rheumatoid arthritis (RA) have elevated Epstein-Barr virus (EBV) load in their peripheral blood mononuclear cells (PBMCs) and whether it is correlated with the HLA-DR genes they express, we developed an accurate EBV DNA quantitative assay using real-time polymerase chain reaction (PCR) with fluorescent probes. METHODS: We studied the EBV DNA load in the PBMCs of 84 patients with RA, 69 normal controls, and 22 patients with rheumatic conditions other than RA. A 214-bp segment from the long internal repeat of EBV was amplified from 500 ng of PBMC DNA (150,000 cells) and quantified by real-time PCR with fluorescent probes. RESULTS: We demonstrated that in patients with RA, the EBV DNA load in PBMCs is increased almost 10-fold compared with that in normal controls. The EBV load is stable over time and is not obviously influenced by disease-modifying antirheumatic drugs or HLA-DR. CONCLUSION: Patients with RA have elevated EBV load in their peripheral blood.

Arthritis, Rheumatoid↗

Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment.

VACUTAINER PPT plasma preparation tubes were evaluated to determine the effects of various handling and shipping conditions on plasma human immunodeficiency virus (HIV) load determinations. Plasmas obtained from PPT tubes stored and shipped under nine different conditions were compared to conventional EDTA tube plasmas stored at -70 degrees C within 2 h after phlebotomy. Compared to viral loads in frozen EDTA plasma, those in PPT tube plasma that was frozen immediately and either separated or shipped in situ were not significantly different. Viral loads in PPT tube plasma after storage for 6 h at either room temperature or 4 degrees C, followed by shipment at ambient temperature or on wet or dry ice, were not significantly different from baseline viral loads in EDTA or PPT plasma. The results of this study indicate that the HIV load in PPT tube plasma is equivalent to that in standard EDTA plasma. Plasma viral load is not affected by storage or shipment temperature when plasma is collected in PPT tubes. Furthermore, plasmas can be shipped in spun PPT tubes, and the tubes provide a safer and more convenient method for sample collection and transport than regular EDTA tubes.

Anticoagulants↗

Human immunodeficiency virus type 1 hypersusceptibility to amprenavir in vitro can be associated with virus load response to treatment in vivo.

The human immunodeficiency virus type 1 protease mutation N88S, which is occasionally selected by treatment with nelfinavir or indinavir, confers hypersusceptibility to amprenavir in vitro. The clinical relevance of this observation is unclear. We report a case of N88S developing after virologic failure of both indinavir- and nelfinavir-containing regimens that was managed successfully with a regimen that contained amprenavir.

Adult↗

Outcome of perinatal hepatitis B virus exposure is dependent on maternal virus load.

To evaluate the role of maternal hepatitis B virus (HBV) DNA levels in perinatal infection, two nested case-control studies were done within a cohort of 773 hepatitis B surface antigen (HBsAg)-positive Taiwanese women and their infants. As serum HBV DNA levels increased from < 0.005 to > or = 1.4 ng/mL among the hepatitis B e antigen (HBeAg)-positive mothers, the odds ratio (OR) for having a persistently infected infant increased from 1.0 to 147.0 (P for trend < .001). Among HBeAg-negative mothers, the OR for having a persistently infected infant was 19.2 (95% confidence interval, 2.3-176.6) in mothers with high versus low levels of serum HBV DNA. A logistic regression analysis identified maternal HBV DNA to be a stronger independent predictor of persistent infection than HBeAg status. Thus, perinatal exposure to high levels of maternal HBV DNA is the most important determinant of infection outcome in the infant.

Case-Control Studies↗

Virus load and neuropathology in the FIV model.

The FIV (feline immunodeficiency virus) induces in cats brain changes presenting similarities with those observed in human immunodeficiency virus infection. This FIV model was used to study the relationship between viral load in brain, in lymphoid organs and central nervous system (CNS) changes during the early and late stages of infection. Early brain changes were analyzed in animals experimentally infected with two different FIV isolates and sacrificed at 7 and 15 days, 1, 2, 6, and 12 months post inoculation (p.i.). Late CNS abnormalities were analyzed in naturally FIV-infected cats referred to the Veterinary School of Nantes. For each animal, one cerebral hemisphere was fixed and examined using routine techniques. The characterization of FIV replicating cells by in situ hybridization was performed on the other half frozen hemisphere on sections performed in the anterior and the median regions of the brain. During the early stages of infection, moderate gliosis with glial nodules and sometimes white matter pallor and meningitis were associated with few infected cells scattered in the brain. Infection was an early event as infected cells could be detected in brain at 7 p.i. For each cat, these findings were found identical in the two analyzed areas. During the late stages, brain lesions and the number of virus replicating cells increased especially in animals with perivascular infiltrates. The multinucleated giant cells encephalitis was never observed and the number of FIV replicating cells scattered in the whole brain was always low. This discrepancy between the number of replicating cells and the brain lesions, corroborates the hypotheses suggesting that brain injuries may be mediated via diffusive factors and amplification processes through cytokine cascades and cell activations.

Animals↗

Monitoring and modulation of Epstein-Barr virus loads in pediatric transplant patients.

A major risk faced by bone-marrow and solid organ transplant patients is the development of post-transplant lymphoproliferative disease or post-transplant lymphoma (PTLD). In pediatric transplantation, PTLD onset is often associated with a rapid rise in Epstein-Barr virus (EBV) load in peripheral blood mononuclear cells (PBMC). We have analyzed EBV viral loads in PBMC over time using real-time quantitative PCR in 56 patients, 19 of which have been followed for more than 1 year. In nine patients; eight bone marrow (BMT) and one kidney transplant, PTLD was associated with a rapid rise in viral load, exceeding 1 x 10(5) EBV genomes/microg of PBMC-derived DNA. Four of these patients exceeded 1 x 10(6) EBV genomes/microg PBMC DNA. All patients with viral loads exceeding 1 x 10(5) EBV genomes/microg PBMC DNA were clearly at high risk for transplant-associated mortality, with only six of nine surviving. Importantly, only one of these deaths was directly attributable to EBV. A second elevated state of EBV load, defined as exceeding 2 x 10(4) EBV genomes/microg PBMC, was seen in a total of 12 BMT, kidney, heart, and liver transplant patients. These patients did not appear to be at immediate lethal risk for PTLD and one EBV-attributable death was found in this group as well. Thirty-four transplant patients whose EBV viral load oscillated from undetectable to 10 000 EBV genomes/microg PBMC DNA are reported as well. The threshold for normal EBV viral load based on our combined experience with viral load analysis is defined as 1 x 10(4) EBV genomes/microg PBMC DNA. The ability to rapidly analyze EBV load allows rapid changes in viral load, such as those that occur with PTLD onset, and the impact of anti-CD20 antibody therapy to be rapidly detected.

Bone Marrow Transplantation↗

High JC virus load in gastric cancer and adjacent non-cancerous mucosa.

The JC virus (JCV) infects a large proportion of the worldwide population and approximately 90% of adults are seropositive. Recent reports have described the possibility of its oncogenetic role in several malignancies. The aim of the present study was to assess the oncogenetic significance of JCV for gastric cancer. Twenty-two sample pairs of fresh tumor and adjacent non-cancerous tissue (ANCT) as well as 10 normal gastric mucosa specimens were investigated on the basis of nested polymerase chain reaction (PCR) followed by Southern blotting, DNA direct sequencing, real-time PCR, in situ PCR and immunohistochemistry. The T antigen sequence was detected in 86.4% of gastric cancers and ANCT, and in 100% of the normal mucosa samples, as for virus capsid protein, 54.1%, 68.1% and 70%, respectively. A generally low incidence was noted for agnoprotein. The JCV DNA load was approximately 10-fold higher in both gastric cancers and paired ANCT (4784 +/- 759 and 5394 +/- 1466 copies/microg DNA, respectively) than in normal gastric tissue (542.4 +/- 476.0 copies/microg DNA, P < 0.0001). In situ PCR revealed sporadic JCV genome-positive cancer cells and foveolar epithelial cells. T antigen protein expression assessed by immunohistochemistry was detected only in one case (1/22; 4.5%), probably because the half life of T antigen might be short. It was concluded that the gastric epithelium in most Japanese people is infected with JCV at a low rate but levels of infection are increased markedly in both cancer cells and ANCT, indicating that multiplication of JCV copies might be a risk factor and a background for gastric carcinogenesis.

Aged↗

Cidofovir in AIDS-associated progressive multifocal leukoencephalopathy: a monocenter observational study with clinical and JC virus load monitoring.

A monocenter observational study was conducted to determine the clinical and virological effects of cidofovir added to highly active anti-retroviral therapy (HAART) in AIDS-associated progressive multifocal leukoencephalopathy (PML). Exposure to other anti-viral drugs or late initiation of cidofovir were exclusion criteria. Of the 53 consecutive patients with virologically proven PML admitted at the NeuroAIDS Unit of Bicêtre Hospital between May 1996 and July 2000 and having received HAART with or without cidofovir, 46 met the inclusion criteria. Cidofovir was initiated in most cases on compassionate grounds. The 22 patients treated with HAART only (HAART group) were compared to the 24 patients treated with HAART and cidofovir (CDV group). Survival, neurological outcome assessed by the expanded disability status scale (EDSS), and monitoring of the JC virus (JCV) load in CSF were investigated prospectively. At baseline (date of initiation or intensification of HAART), both groups were similar regarding CD4 cell count, plasma HIV load, CSF JCV load, EDSS, and demographic features. Both groups had similar response to HAART in terms of plasma HIV load and CD4 cell count. At month 6, CSF-JCV load was below the detection level in 8 out of 24 (33%) patients from the CDV group and 7 out of 18 (39%) patients from the HAART group (P = 0.71). One-year cumulative probability of being alive was 62% in the CDV group and 53% in the HAART group (P = 0.72). However, an additional benefit with respect to survival was observed in patients who were given cidofovir after adjustment to the following baseline variables (CSF-JCV load, CD4 cell count, and EDSS). Despite the addition of cidofovir to HAART, no significant benefit had been observed in neurological outcome, particularly in patients with an early worsening.

AIDS-Related Opportunistic Infections↗

Distribution of hepatitis C virus (HCV) RNA in whole blood and blood cell fractions: plasma HCV RNA analysis underestimates circulating virus load.

Previous experiments using a cationic surfactant to detect hepatitis C virus (HCV) RNA in whole blood (WB) suggested that WB was a more plentiful source of viral RNA than was plasma. The relative HCV RNA titers in WB, plasma, peripheral blood mononuclear cells (PBMC), neutrophils, and red blood cells (RBC)/platelets from 10 patients with chronic HCV infection were compared. WB contained significantly more HCV RNA than plasma, which contained more HCV RNA than PBMC, neutrophils, or RBC/platelets (P < .001). To determine if this increased sensitivity was clinically relevant, results of WB and plasma HCV RNA assays were compared with commercial quantitative and qualitative plasma HCV RNA assay results obtained for patients receiving interferon therapy. WB was significantly more sensitive than commercial plasma reverse transcription-polymerase chain reaction for detecting HCV RNA (P < .005). These data indicate that a significant proportion of HCV RNA in peripheral blood is not identified by standard plasma RNA detection methods.

Blood Cells↗

A longitudinal and prospective study of Epstein-Barr virus load in AIDS-related non-Hodgkin lymphoma.

BACKGROUND: Epstein-Barr virus (EBV) may be causally associated with non-Hodgkin Lymphoma (NHL) in HIV-infected patients. OBJECTIVES: To compare EBV load in whole blood in AIDS-NHL patients, HIV non-AIDS patients and non-HIV-infected persons, and to prospectively measure EBV load in whole blood in AIDS-NHL patients. STUDY DESIGN: Longitudinal and prospective study. RESULTS: We observed no statistical difference in EBV load between AIDS-NHL (3.69log(10) copies/mL [interquartile range (IQR): 2.89-4.27]) and HIV non-AIDS patients (3.08log(10) copies/mL [IQR: 1.29-3.57]) but AIDS-NHL patients had significantly higher EBV loads than HIV-negative controls (1.19log(10) copies/mL [IQR: 0.00-3.29]). We noticed an inverse correlation between CD4+ lymphocytes count and EBV load in patients with AIDS-NHL (r(2)=0.41, P=0.01). In the longitudinal study, the mean EBV load three months after NHL diagnosis decreased significantly (mean difference=-1.69log(10) copies/mL [95% confidence interval: -0.32; -3.04]; P=0.03) under chemotherapy but was still elevated in patients with relapses or no response to chemotherapy. CONCLUSION: Although EBV load seems a suboptimal marker for the diagnosis of AIDS-NHL, we observed a significant decrease of EBV load in patients treated with chemotherapy and a strong association between NHL outcome and EBV load in whole blood.

Acquired Immunodeficiency Syndrome↗

Biological dynamics of hepatitis B virus load in dialysis population.

BACKGROUND: Control of the spread of hepatitis B virus (HBV) infection in dialysis units has been one of major advances in the management of end-stage renal disease. However, the natural history of HBV in dialysis patients remains unclear. The aim of this study is to measure monthly HBV viral load (HBV DNA) in a large cohort (n = 29) of hepatitis B surface antigen (HBsAg)-positive chronic dialysis patients during 12 months. METHODS: HBV DNA was measured using the Amplicor HBV Monitor Test (Roche Diagnostics, Branchburg, NJ), an in vitro assay using polymerase chain reaction nucleic acid amplification and DNA hybridization for the quantitative measurement of HBV DNA in serum. RESULTS: We observed three HBV DNA patterns: (1) patients persistently positive by Amplicor HBV Monitor Test (persistent HBV DNA; 7 of 29 patients; 24.1%), (2) individuals with alternatively positive and negative results (intermittent HBV DNA; 18 of 29 patients; 62.1%), and (3) patients persistently negative by Amplicor HBV Monitor Test (4 of 29 patients; 13.8%). HBV viral load was greater in patients with persistent compared with intermittent HBV DNA (persistently HBV DNA positive; 2.686 x 10(4) copies/mL; 95% confidence interval [CI], 5.2499 x 10(4) to 1.8158 x 10(4)copies/mL) versus intermittently HBV DNA positive (1.071 x 10(3) copies/mL; 95% CI, 8.524 x 10(3) to 4.09 x 10(2) copies/mL; P = 0.0001). In the entire group, HBV load at study entry was low and did not change versus the end of follow-up. CONCLUSION: Three patterns of HBV viremia in dialysis patients over time were assessed; HBV load was not high and was relatively stable. HBsAg-positive patients who were intermittently HBV DNA positive had less HBV viral load than persistently HBV DNA-positive patients. Periodic testing for HBV DNA to assess the virological status of HBsAg-positive dialysis patients is recommended.

Aged↗

Exposure to holoendemic malaria results in elevated Epstein-Barr virus loads in children.

Perennial and intense malaria transmission (holoendemic malaria) and Epstein-Barr virus (EBV) infection are 2 cofactors in the pathogenesis of endemic Burkitt lymphoma (eBL). In the present study, we compared EBV loads in children living in 2 regions of Kenya with differing malaria transmission intensities: Kisumu District, where malaria transmission is holoendemic, and Nandi District, where malaria transmission is sporadic. For comparison, blood samples were also obtained from US adults, Kenyan adults, and patients with eBL. Extraction of DNA from blood and quantification by polymerase chain reaction give an EBV load estimate that reflects the number of EBV-infected B cells. We observed a significant linear trend in mean EBV load, with the lowest EBV load detected in US adults and increasing EBV loads detected in Kenyan adults, Nandi children, Kisumu children, and patients with eBL, respectively. In addition, EBV loads were significantly higher in Kisumu children 1-4 years of age than in Nandi children of the same age. Our results support the hypothesis that repeated malaria infections in very young children modulate the persistence of EBV and increase the risk for the development of eBL.

Adolescent↗

HLA-B63 presents HLA-B57/B58-restricted cytotoxic T-lymphocyte epitopes and is associated with low human immunodeficiency virus load.

Several HLA class I alleles have been associated with slow human immunodeficiency virus (HIV) disease progression, supporting the important role HLA class I-restricted cytotoxic T lymphocytes (CTL) play in controlling HIV infection. HLA-B63, the serological marker for the closely related HLA-B*1516 and HLA-B*1517 alleles, shares the epitope binding motif of HLA-B57 and HLA-B58, two alleles that have been associated with slow HIV disease progression. We investigated whether HIV-infected individuals who express HLA-B63 generate CTL responses that are comparable in breadth and specificity to those of HLA-B57/58-positive subjects and whether HLA-B63-positive individuals would also present with lower viral set points than the general population. The data show that HLA-B63-positive individuals indeed mounted responses to previously identified HLA-B57-restricted epitopes as well as towards novel, HLA-B63-restricted CTL targets that, in turn, can be presented by HLA-B57 and HLA-B58. HLA-B63-positive subjects generated these responses early in acute HIV infection and were able to control HIV replication in the absence of antiretroviral treatment with a median viral load of 3,280 RNA copies/ml. The data support an important role of the presented epitope in mediating relative control of HIV replication and help to better define immune correlates of controlled HIV infection.

Amino Acid Sequence↗

Correlation between serum transaminase activity and virus load among patients with chronic liver disease type B.

Newly developed hepatitis B virus (HBV)-DNA quantitative assays, transcription-mediated amplification and hybridization protection assay (TMA-HPA) and branched-DNA assay were clinically evaluated. The subjects consisted of 160 chronic HBV carriers; 48 were hepatitis Be antigen (HBeAg)-positive, whereas 109 were anti-HBe-positive (three were both negative). All subjects with HBeAg, except one, showed high HBV-DNA replication levels (>/=10(5.8) copies/ml). In HBeAg negative subjects, there was a strong correlation between the serum HBV-DNA and alanine aminotransferase (ALT) levels; ALT level was usually normal if the samples tested showed an HBV-DNA level less than 10(5)/ml, whereas, the majority of the sera with an HBV-DNA concentration greater than 10(7)copies/ml showed elevation in serum ALT level. An intermediate range of HBV-DNA level (10(5)-10(7) copies/ml) was associated with variable ALT activity. In conclusion, a serum HBV-DNA level associated with ALT elevation was lower in patients with type B chronic liver disease negative for HBeAg compared with their HBeAg-positive counterparts. There was usually no or mild liver disease activity when patients with chronic HBV infection have serum HBV-DNA levels less than 10(5)copies/ml.

Journal Article↗

Vitamin A supplementation and human immunodeficiency virus load in injection drug users.

The use of vitamin A therapy during human immunodeficiency virus (HIV) infection is under clinical investigation, and vitamin A could potentially modulate HIV replication because the virus genome contains a retinoic acid response element. A randomized, double-masked, placebo-controlled clinical trial was conducted to determine the impact of single high-dose vitamin A supplementation, 60-mg retinol equivalent (200,000 IU), on HIV load and CD4 lymphocyte count. HIV-infected injection drug users (120) were randomly allocated to receive vitamin A or placebo. Plasma vitamin A level, CD4 lymphocyte count, and HIV load were measured at baseline and 2 and 4 weeks after treatment. Vitamin A supplementation had no significant impact on HIV load or CD4 lymphocyte count at 2 and 4 weeks after treatment. This study suggests that high-dose vitamin A supplementation does not influence HIV load.

Adult↗

Decreasing the Epstein-Barr virus load by adjusting the FK506 blood level.

To prevent post-transplant lymphoproliferative disease (PTLD), the viral load must be diminished before the symptoms of Epstein-Barr virus (EBV) infection appear. Twenty-three consecutive liver transplant recipients were entered into our study to identify the characteristics of post-transplant EBV-infected patients and to clarify the correlation between the FK506 blood level and EBV load. After transplantation, EBV-DNA appeared more frequently in patients who had been seronegative before transplantation than in seropositive patients (10/13 versus 1/10; P=0.0014). As for rejection, resistance to steroid pulse therapy, and FK506 trough level, there were no significant differences between patients with and without EBV infection. In patients with primary EBV infection after transplantation, there was a strong correlation ( r=0.681) between the FK506 level and the viral load. In liver transplant recipients, especially in those seronegative for EBV, it is necessary to check the viral load by polymerase chain reaction (PCR) carefully after liver transplantation, before any symptom appears.

Adolescent↗

Varicella-zoster virus load and cochleovestibular symptoms in Ramsay Hunt syndrome.

OBJECTIVES: The mechanism by which varicella-zoster virus (VZV) reactivation causes cochleovestibular symptoms (CVSs) in patients with Ramsay Hunt syndrome (RHS) remains to be elucidated. The present study analyzed the relationship between VZV load and the onset of CVSs in RHS. METHODS: The subjects consisted of 56 patients with RHS; 29 exhibited CVSs and facial paralysis (FP; group 1), and 27 exhibited FP without CVSs (group 2). The VZV DNA copy number in the saliva was measured with a quantitative polymerase chain reaction. Anti-VZV antibodies were assayed by an enzyme-linked immunosorbent assay with paired sera. RESULTS: There was no significant difference in maximum viral copy number between the two groups. In group 1, CVSs occurred at various times between the early phase and the regression phase of VZV reactivation. In some patients, CVSs occurred in the early phase of VZV reactivation, before the onset of zoster lesions and FP. CONCLUSIONS: There are various different patterns in the development of eighth cranial nerve dysfunction, which is caused by progression of neuritis or labyrinthitis following VZV reactivation. Our data suggest that CVSs in RHS may also be caused by reactivation of VZV in the spiral and/or vestibular ganglia.

Antibodies, Viral↗