Comparison of two highly automated DNA extraction systems for quantifying Epstein-Barr virus in whole blood

BACKGROUND: Optimal automated molecular methods are needed to monitor Epstein-Barr virus (EBV) infections in transplant recipients. OBJECTIVES: To compare the extraction of EBV DNA from whole blood using the COBAS Ampliprep and the MagNA Pure instruments (Roche) for quantifying EBV DNA by real-time PCR. STUDY DESIGN: EBV DNA content was determined on clinical samples extracted by both systems. RESULTS: The detection limit was 2.16log(10)copies/mL using the COBAS Ampliprep extraction system. Specificity was 100% and we saw no cross-contamination. Extraction was linear from 2.60 to 5.60log(10)copies/mL. The intra-assay variation was 1.91% for 3.60, 2% for 4.60 and 4.51% for 5.60log(10)copies/mL; inter-assay variation was 4.88%. Sixty-six samples were tested: 26 were positive and 28 were negative by both methods. One sample was MagNA Pure positive/COBAS Ampliprep negative (virus load 3.15log(10)copies/mL) and 10 samples were MagNA Pure negative/COBAS Ampliprep positive (virus loads from 1.59 to 3.51log(10)copies/mL) (P<0.0001). Both methods gave similar quantitative results (average difference 0.07log(10)copies/mL) which were well correlated (r=0.73, P<0.001). CONCLUSIONS: The COBAS Ampliprep extraction system is comparable to the MagNA Pure and offers a high reliability for extracting EBV DNA from whole blood.     

Whole blood real-time quantitative PCR for cytomegalovirus infection follow-up in transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) remains a major opportunistic agent among transplant recipients. While detection of CMV pp65-lower matrix protein (pp65Ag) is still widely used for monitoring CMV infection, real-time PCR assays have been recently developed for routine quantitation of CMV DNA. However, correlations are lacking between results of pp65Ag and quantitative PCR assays and there is no consensus yet as to the more appropriate blood compartment (whole blood (WB), leukocytes, plasma) to be tested with PCR assays. OBJECTIVES: The aims of the study were to determine, in a population of transplant recipients: (i) the correlation between pp65Ag and CMV quantitative real-time PCR in our setting and (ii) the utility of plasma CMV DNA quantitation in comparison to WB quantitation. METHODS: In 170 blood samples (from 61 solid organ or bone marrow transplant recipients) with pp65Ag results, CMV quantitation was performed in WB and plasma using an in-house real-time quantitative PCR. RESULTS: Real-time PCR and pp65Ag results in WB were correlated: thresholds of 10 and 50(+) cells/200,000 cells were equivalent to 3.3 log(10)copies/mL (2,000 copies/mL) and 3.8 log(10)copies/mL (6,300 copies/mL), respectively. When WB viral load was >or=3.6 log(10)copies/mL, the risk to have a negative plasma CMV DNA result was 相似文献   

Torquetenovirus Dynamics and Immune Marker Properties in Patients Following Allogeneic Hematopoietic Stem Cell Transplantation: A Prospective Longitudinal Study

Torquetenovirus (TTV) has been proposed as a marker of immune function in patients receiving immunosuppression after solid organ transplantation. This study aimed to define TTV plasma dynamics and investigate clinical associations in patients following allogeneic hematopoietic stem cell transplantation (HSCT). This was a single-center prospective longitudinal study involving 50 consecutive patients treated with HSCT between March 2015 and April 2016. TTV plasma DNA levels were measured with quantitative PCR at 12 consecutive time points during the first year after HSCT. Forty of the 50 patients (80%) had detectable TTV viremia before HSCT (median level, 5.37 log10 copies/mL; interquartile range [IQR], 3.51-6.44 log10 copies/mL). All patients subsequently developed TTV viremia during the follow-up period. Plasma viral loads evolved dynamically over time, with a peak of 8.32 log10 copies/mL (IQR, 7.33-9.35 log10 copies/mL) occurring at 79 days (IQR, 50-117 days) following HSCT and a stable plateau toward the end of the follow-up period. The type of malignancy, the use of antithymocyte globulin during conditioning, and the occurrence of acute graft-versus-host disease requiring systemic therapy had temporary effects on TTV dynamics. TTV levels showed a significant correlation with absolute lymphocyte counts following engraftment (r_s = -.27; P?<?.01) and with cytomegalovirus (CMV; r_s=.39; P?<?.01) and Epstein-Barr virus (EBV; r_s=.45; P?=?.02) viral loads during phases of viremia. Immune-related clinical events were not predicted by TTV levels. TTV viremia occurred universally and was sustained throughout the first year after HSCT. Several variables and events before and after HSCT were correlated with TTV levels and hint toward immune marker properties of TTV, but their complex interactions might perturb the capability of TTV to predict immune-related complications in this population.     

Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4.5 x 10(6) copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative BamHI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for beta-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation.     

A virologic pilot study of valacyclovir in infectious mononucleosis.

BACKGROUND: Infectious mononucleosis decreases the productivity of many college students and Epstein-Barr virus (EBV) infection may result in long-term immune damage. OBJECTIVES: Evaluate the antiviral effect of valacyclovir during EBV-related acute infectious mononucleosis and explore potential clinical benefits. STUDY DESIGN: University students who presented during the first 7 days of illness were randomized to receive valacyclovir 3g/day for 14 days or not. The quantity of Epstein-Barr virus (EBV) DNA in oral and whole blood samples was determined by real-time (TaqMan) PCR. The primary outcome was the proportion of subjects with laboratory-confirmed primary EBV infection who had >or=2 log10 decrease in EBV copies/mL in oral washes during the treatment period. Secondary outcomes included clinical effects. RESULTS: Twenty subjects were studied. The proportion of valacyclovir recipients versus control subjects who had >or=2 log10 decrease in EBV copies was significantly greater for both oral wash fluid-derived cell pellet (P=0.03) and supernatant (P=0.001) samples. At the end of the treatment period, the number of reported symptoms (P=0.03) and the severity of illness (P=0.049) were reduced among valacyclovir recipients as compared with controls. CONCLUSIONS: Valacyclovir therapy caused a reduction of EBV excretion and possibly produced a clinical benefit in infectious mononucleosis. Because our study was small and not placebo-controlled, these results must be confirmed by a larger, placebo-controlled trial.     

Quantitation of BK virus DNA for diagnosis of BK virus-associated nephropathy in renal transplant recipients

Quantitative measurement of BK virus DNA (Q-BKDNA) has been used for the early diagnosis and monitoring of BK virus-associated nephropathy (BKVAN). This study was designed to determine the BKDNA cutoff for the diagnosis of BKVAN. Between June 2005 and February 2007, 64 renal transplant recipients taken renal biopsies due to renal impairment submitted plasma and urine for Q-BKDNA. Eight BKVAN patients (12.5%) had median viral loads of 6.0 log10 copies/mL in plasma and 7.3 log10 copies/mL in urine. Among 56 non-BKVAN patients, 45 were negative for Q-BKDNA; 4 were positive in plasma with a median viral load of 4.8 log10 copies/ mL, and 10 were positive in urine with a median viral load of 4.8 log10 copies/mL. Receiver operating characteristic curve analysis showed that a cutoff of 4.5 log10 copies/mL in plasma and a cutoff of 5.9 log10 copies/mL in urine had a sensitivity of 100% and a specificity of 96.4%, respectively. A combined cutoffs of 4 log10 copies/ mL in plasma and 6 log10 copies/mL in urine had better performance with a sensitivity of 100% and a specificity of 98.2% than each cutoff of urine or plasma. QBKDNA with the combined cutoffs could reliably diagnose BKVAN in renal transplant recipients.     

Automated extraction of viral-pathogen RNA and DNA for high-throughput quantitative real-time PCR

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples (hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTA-blood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 +/- 0.06 for HCV and 0.97 +/- 0.03 for HBV, indicating a linear extraction from 100 to 1.0 x 10(5) HCV IU/ml and from 100 to 1.0 x 10(6) HBV IU/ml. Intra- and interrun variability was below 0.23 log(10) IU/ml for 2.98 to 5.28 log(10) HCV IU/ml and 2.70 to 5.20 log(10) HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log(10) HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log(10) HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log(10) copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log(10) copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.     

Prevalence and risk factors for developing K65R mutations among HIV-1 infected patients who fail an initial regimen of fixed-dose combination of stavudine, lamivudine, and nevirapine.

BACKGROUND: A fixed-dose combination of stavudine, lamivudine, and nevirapine (d4T/3TC/NVP) is extensively used as initial antiretroviral regimen in developing countries. K65R mutations that occur after failing this regimen prevent the use of tenofovir, didanosine, and abcavir in the second-line regimen. OBJECTIVES: To determine the prevalence and risk factors of K65R mutations after failing d4T/3TC/NVP. STUDY DESIGN: Genotypic resistance testing was conducted among HIV-1 infected patients who experienced virological failure with an initial regimen of d4T/3TC/NVP. RESULTS: There were 122 patients who received antiretroviral therapy (ART) for a median (IQR) duration of 19 (13-27) months. Median (IQR) CD4 cell count and plasma HIV-1 RNA at virological failure was 174 (109-264) cells/mm(3) and 4.0 (3.7-4.6)log copies/mL, respectively. The prevalence of K65R mutations was 7%. Patients with K65R mutations had higher plasma HIV-1 RNA at virological failure compared to patients without K65R mutations (4.9log copies/mL vs. 4.0log copies/mL, p=0.001). By logistic regression analysis only plasma HIV-1 RNA at failure correlated with the occurrence of K65R mutations [OR 4.2 (95% CI, 1.5-11.2) per 0.5log copies/mL increment of HIV-1 RNA]. CONCLUSIONS: Seven percent of patients had K65R mutations after failing an initial d4T/3TC/NVP regimen. Tenofovir, didanosine, and abcavir cannot be used in second-line regimen for these patients. HIV-1 RNA at the time that virological failure was detected correlated with the occurrence of K65R mutations.     

Association between paired plasma and cervicovaginal lavage fluid HIV-1 RNA levels during 36 months

OBJECTIVE: To determine the patterns and predictors of genital tract HIV-1 RNA levels during a 36-month period. METHODS: HIV-1 RNA levels were measured blood in plasma and the genital tract (by cervicovaginal lavage [CVL]) at baseline before highly, active antiretroviral therapy, at 2 and 4 weeks and every 6 months. Viral loads were measured using nucleic acid sequence-based amplification assay with a lower limit of detection of 2.6 log10 copies/mL. RESULTS: Ninety-seven women had a median of 30.4 months' follow-up, with 530 paired PVL and CVL specimens. The strongest predictor of CVL fluid HIV-1 RNA detection was PVL of more than 2.6 log10 copies/mL, with an odds ratio of 13.7 (P < 0.0001). Each log10 unit increase in PVL increased the odds of detecting HIV-1 RNA in CVL fluid by 2.6 folds (P = 0.0002). Cervicovaginal lavage fluid HIV-1 RNA exceeded PVL on 5% of visits. When patients achieved undetectable levels of HIV-1 RNA in both plasma and CVL fluid, rebound of HIV-1 RNA occurred in plasma first or concurrently with CVL fluid HIV-1 RNA. CONCLUSIONS: Plasma viral load is the strongest predictor of CVL fluid HIV-1 RNA detection. Cervicovaginal lavage fluid HIV-1 RNA levels are generally lower than PVL. Plasma viral load is more likely to rebound first or at the same time as CVL fluid viral load.     

Monitoring of Epstein-Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease is a life-threatening complication following hematopoietic stem cell transplantation. A quantitative polymerase chain reaction to evaluate EBV-genome copy numbers based on a nested polymerase chain reaction and an end-point dilution was used. Applying this assay EBV load was prospectively screened weekly in 123 patients after transplantation. The results demonstrate that EBV reactivations with more than 1,000 EBV-genome copies measured in 10(5) peripheral blood mononuclear cells were observed in 31 patients (25.2%). Three patients developed lymphoproliferative disease with extremely high EBV-genome copies in peripheral blood mononuclear cells (>100,000 copies/10(5) cells) and plasma. After combined antiviral and immune therapy two of three patients showed a dramatic decrease of EBV load and survived, while the third patient died of lymphoma. A subclinical EBV reactivation was observed in 24 cases (19.5%) with EBV-genome copies in 10(5) peripheral blood mononuclear cells ranging between 2,500 and mostly 10,000. After reduction of immunosuppression the EBV levels normalized. In four patients, the high copy number of > or =80,000 copies/10(5) peripheral blood mononuclear cells and plasma positivity prompted us to start pre-emptive therapy with rituximab and cidofovir for prevention of lymphoproliferative disease. After drug administration the high EBV load was reduced remarkably. Ninety-two patients (74.8%) who had < or =1,000 copies/10(5) peripheral blood mononuclear cells did not develop EBV-associated lymphoproliferative disease. In conclusion, monitoring of EBV load is a sensitive and useful parameter in the surveillance of EBV reactivation for early intervention in EBV-associated lymphoproliferative disease as well as for follow-up of the efficacy of therapy.     

Dried blood spots versus plasma for the quantitation of HIV-1 RNA using a real-Time PCR, m2000rt assay

High costs and stringent requirements for storage and transport of plasma, often prohibit the availability of HIV viral load quantification in resource-limited settings. Dried blood spots (DBS) represent a better method of specimen collection that removes many of these logistical and technical limitations. The present study aimed to assess the performance of the Abbott m2000rt assay for quantitation of HIV-1 RNA in DBS specimens using plasma as a "gold standard" for comparison. One hundred paired DBS and plasma specimens were collected from patients infected with HIV, who were 18 years and older during routine visits to a private tertiary-care clinic in Chennai, India. HIV-1 RNA was extracted manually and then detected using the m2000rt assay. The mean plasma and DBS viral loads were 4.27 (95% CI: 2.65, 5.88) and 4.14 (95% CI: 1.96, 6.32) log copies/mL, respectively. The overall sensitivity of DBS reached 95%; with sensitivities of 62%, 88% and 100% when stratified by viral load ranges of ≤1000, 1000-3000 and >3000 copies/mL, respectively. An over quantitation of the viral load with DBS was observed in pairs with plasma viral load<3000 copies/mL [d=-0.3 log copies/mL (ranging from -0.1 to 0.6 log copies/mL)]. The study showed a strong concordance in RNA levels between plasma and DBS. The use of DBS specimens should be considered for HIV monitoring and for detection of virologic failure in resource-limited settings.     

Antiretroviral Genotypic Resistance Mutations in HIV-1 Infected Korean Patients with Virologic Failure

Resistance assays are useful in guiding decisions for patients experiencing virologic failure (VF) during highly-active antiretroviral therapy (HAART). We investigated antiretroviral resistance mutations in 41 Korean human immunodeficiency virus type 1 (HIV-1) infected patients with VF and observed immunologic/virologic response 6 months after HAART regimen change. Mean HAART duration prior to resistance assay was 45.3±27.5 months and commonly prescribed HAART regimens were zidovudine/lamivudine/nelfinavir (22.0%) and zidovudine/lamivudine/efavirenz (19.5%). Forty patients (97.6%) revealed intermediate to high-level resistance to equal or more than 2 antiretroviral drugs among prescribed HAART regimen. M184V/I mutation was observed in 36 patients (87.7%) followed by T215Y/F (41.5%) and M46I/L (34%). Six months after resistance assay and HAART regimen change, median CD4+ T cell count increased from 168 cells/µL (interquartile range [IQR], 62-253) to 276 cells/µL (IQR, 153-381) and log viral load decreased from 4.65 copies/mL (IQR, 4.18-5.00) to 1.91 copies/mL (IQR, 1.10-3.60) (P<0.001 for both values). The number of patients who accomplished viral load <400 copies/mL was 26 (63.4%) at 6 months follow-up. In conclusion, many Korean HIV-1 infected patients with VF are harboring strains with multiple resistance mutations and immunologic/virologic parameters are improved significantly after genotypic resistance assay and HAART regimen change.     

Suppressive acyclovir therapy reduces HIV cervicovaginal shedding in HIV- and HSV-2-infected women, Chiang Rai, Thailand

BACKGROUND: Herpes simplex virus type 2 infection is important in the HIV epidemic and may contribute to increased HIV transmission. We evaluated the effect of suppressive acyclovir therapy on cervicovaginal HIV-1 shedding. METHODS: HIV-1- and herpes simplex virus type 2-coinfected women aged 18-49 years with CD4 counts >200 cells/microL were enrolled in a randomized crossover trial of suppressive acyclovir therapy (NCT00362596, http://www.clinicaltrials.gov). For each woman, monthly plasma and weekly cervicovaginal lavage specimens were collected; the mean of the monthly median cervicovaginal lavage HIV-1 viral load and plasma HIV-1 viral load was compared. RESULTS: Sixty-seven women were enrolled; at baseline, median CD4 count was 366 cells/microL, and median HIV-1 plasma viral load was 4.6 log10 copies/mL. The mean cervicovaginal lavage HIV-1 viral load was 1.9 (SD 0.8) log10 copies/mL during the acyclovir month and 2.2 (SD 0.7) log10 copies/mL during the placebo month (P < 0.0001); the mean decrease in HIV was 0.3 log10 copies/mL. The mean plasma HIV viral load during the acyclovir month (3.78 log10 copies/mL) was reduced compared with the placebo month (4.26 log10 copies/mL, P < 0.001). CONCLUSIONS: Acyclovir reduced HIV genital shedding and plasma viral load among HIV-1- and herpes simplex virus type 2-coinfected women. Further data from clinical trials will examine the effect of suppressive therapy on HIV transmission.     

CMV plasma DNAemia and risk of cancer among HIV-infected patients: A case–control study nested in the ANRS CO3 Aquitaine Cohort,France, 2002–2007

BackgroundCMV reactivation, which enhances immune senescence, could be associated with a higher risk of cancer.ObjectivesWe compared the prevalence of positive CMV DNAemia in HIV-infected patients with and without cancer.Study designThis case–control study, nested in the ANRS-CO3 Aquitaine Cohort, included patients with a first diagnosis of cancer (2002–2007) as cases. Two controls were matched per case.Cancer risk was estimated using conditional logistic regression models, an Odds Ratio (OR) of 2 could be detected with 80% power. The variables considered were: ≥1 positive CMV DNAemia, CD4+ and CD8+ counts, HIV plasma load. Plasma CMV DNA was retrospectively quantified within the 3-year period preceding the endpoint.ResultsThe 143 cases (93 non-AIDS-related and 50 AIDS-related cancers) and 284 controls had a median age of 47 years (IQR: 41–56). At the time of diagnosis or censorship, for cases and controls, median values were respectively, for CD4+ count: 327 cells/mm³ (IQR: 164–514) and 416 (IQR: 275–582), and for HIV plasma load: 2.6 log₁₀ copies/mL (IQR: 1.7–4.7) and 1.7 log₁₀ copies/mL (IQR: 1.7–3.3). We performed 2056 CMV PCR; 14 cases (9.8% [95% CI: 4.9–14.7]) and 19 controls (6.7% [CI: 3.8–9.6]) presented ≥1 positive PCR. CMV DNAemia was not associated with the risk of cancer (unadjusted and adjusted p-values = 0.19 and 0.54, respectively). HIV load >500 copies/mL was independently associated with a higher risk of cancer (OR = 2.02; p = 0.002; 95% CI: 1.29–3.17).ConclusionThis large case–control study did not show any differential exposure to positive CMV plasma DNAemia between cancer cases and controls.     

Changes in hepatitis C virus (HCV) viral load and interferon-alpha levels in HIV/HCV-coinfected patients treated with highly active antiretroviral therapy

BACKGROUND: Reports are mixed as to whether highly active antiretroviral therapy (HAART) increases liver transaminase levels or hepatitis C virus (HCV) titers in HIV/HCV-coinfected individuals. It is hypothesized that increases in HCV RNA titers may result from changes in endogenous interferon-alpha (IFN-alpha) production. METHODS: HIV/HCV-coinfected patients receiving HAART were tested at baseline, 1, 2, 3, 6, and 9 months for liver transaminase levels, HIV and HCV viral loads, and IFN-alpha. Linear regression analysis was used to determine the effect of HAART on liver transaminase levels, HCV viral load, and IFN-alpha. RESULTS: Initiating HAART did not increase liver transaminase levels in majority of cases. In patients (n = 30) with baseline HIV titer >10,000 copies/mL, HCV titers increased 0.69 log10 and IFN-alpha decreased -0.96 log10 during HAART, in association with a > or =0.5 log10 decrease in HIV titer. As HIV titers reached their nadir approximately 4 months after initiation of HAART, HCV titers remained 0.54 log10 and IFN-alpha -0.71 log10 above and below baseline levels, respectively. HCV titers and IFN-alpha levels did not change from baseline in patients with baseline HIV titer < or =10,000 copies/mL. CONCLUSIONS: Coinfected patients did not have evidence of hepatoxicity HAART. In patients with baseline HIV titer >10,000 copies/mL, suppression of HIV replication by HAART was associated with an increase in HCV titer and a decrease in endogenous IFN-alpha levels.     

Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring in blood has been shown to be essential for the diagnosis of EBV-associated diseases. However, the methods currently used to assess EBV DNA load are often time-consuming and require prior blood separation. OBJECTIVES: The aim of this study was to evaluate the relative diagnostic value of EBV DNA load monitoring in whole blood, peripheral blood mononuclear cells (PBMCs) and plasma after automated DNA extraction using the MagNA Pure extractor followed by LightCycler real-time quantitative PCR (LC-PCR). STUDY DESIGN: First, EBV DNA load was assessed retrospectively after automated or manual extraction on 104 PBMC specimens. Second, EBV DNA load was determined prospectively with the automated extraction procedure in the whole blood, PBMCs and plasma of 100 samples from patients with EBV-related diseases (group 1, n = 20), HIV-seropositive individuals (group 2, n = 66), and healthy EBV carriers (group 3, n = 14). RESULTS: A good correlation was observed between automated and manual extraction on 104 PBMC specimens (r = 0.956; P < 0.0001). In the prospective study, 67 samples were positive in both whole blood and PBMCs, with a good correlation between EBV DNA loads in whole blood and PBMCs (r = 0.936; P < 0.0001). Only 18/100 samples were positive in plasma. Higher viral loads were regularly observed in the three blood compartments from group 1 than from groups 2 and 3. CONCLUSION: This study demonstrated that an automated extraction of EBV DNA is easier to perform in whole blood or plasma than in PBMCs and facilitates the standardisation of EBV DNA measurement by real-time quantitative PCR. The quantitative detection of EBV DNA load in whole blood appeared more sensitive than in plasma for infectious mononucleosis in immunocompetent patients, probably because of a rapid loss of plasmatic EBV DNA. In transplant patients, EBV DNA load monitoring in whole blood and in plasma turned out to be equivalent in terms of feasibility and accuracy for the early diagnosis of post-transplant lymphoproliferative diseases (PTLDs).     

Quantification of Epstein-Barr virus load in Argentinean transplant recipients using real-time PCR.

BACKGROUND: Post-transplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplant recipients that is caused by suppression of T-cell function. OBJECTIVE: Evaluation of the diagnostic value of EBV DNA load in non-fractionated whole blood samples (n = 297) from 110 pediatric transplant patients by real-time PCR. RESULTS AND CONCLUSIONS: Patients with PTLD had a median viral load of 1.08 x 10(5) copies/ml blood (n = 24), which was significantly higher compared with patients without PTLD (median: 50 copies/ml blood, n = 273, P < 0.0001). From receiver operating characteristic (ROC) curve analysis we obtained a cut-off value of 6215 copies/ml blood with a sensitivity of 95.8%, specificity of 71.4%, negative predictive value (NPV) of 99.5% and positive predictive value (PPV) of 22.8%. Thus, real-time PCR proved to be more useful in ruling out than in indicating the presence of PTLD. Further analysis showed that patients without PTLD but developing a post-transplant EBV-primary infection had associated high viral loads that were indistinguishable from those of the PTLD group (statistically not significant). Similarly, the presence of clinical symptoms of disease in patients without PTLD was associated with higher viral loads than in patients that were asymptomatic (P < 0.0001), but the difference was much less significant when compared with the PTLD group of patients (P = 0.0391). These patients who had a high viral load may benefit from a close follow-up of the viral burden.     

Efficacy and safety of a quadruple combination Combivir + abacavir + efavirenz regimen in antiretroviral treatment-naive HIV-1-infected adults: La Francilienne

OBJECTIVE: To evaluate the safety and efficacy of a protease inhibitor sparing, quadruple therapy (Combivir + abacavir + efavirenz) in antiretroviral treatment-naive HIV-1-infected adults. DESIGN: Multicenter open-label pilot study. Clinical and biological assessments were performed at baseline and at weeks 2, 4, 8, 16, 24, 32, 40, 48. RESULTS: Thirty-one subjects enrolled with a median baseline viral load (VL) of 4.69 log10 copies/mL and CD4 cell count of 322 cells/mm3. At week 48, 90% (intention-to-treat [ITT] switch included) and 77% (ITT switch = failure) patients had a VL <50 copies/mL. These results were similar in the population (n = 13) with a VL >100,000 copies/mL at baseline. Combivir + abacavir + efavirenz demonstrated an early antiretroviral response: 58% of patients had plasma HIV-1 RNA <50 copies/mL at week 8. Using a modified assay, the percentage of patients with VL <5 copies/mL at week 48 was 55% (17/31) and 42% (13/31) using ITT (switch included) and ITT (switch = failure), respectively. Median VL decreased by -4.0 log10 copies/mL at week 48 (ITT). Median CD4+ cell count change from baseline at week 48 was +129 cells/mm3 (ITT). Most patients experienced at least one drug-related adverse event that was not considered treatment-limiting by the investigator. There were no cases of abacavir hypersensitivity reactions. CONCLUSIONS: Safety and efficacy results from this study demonstrated that the quadruple regimen Combivir/abacavir/efavirenz is generally safe and displays potent and durable antiretroviral activity in antiretroviral treatment-naive HIV-1-infected patients, offering a promising therapeutic option in a PI-sparing strategy.