Load In Whole Blood Research Articles

Epstein–Barr virus serology and PCR: conflicting results in an immunocompetent host. A case report and review of literature

We present the case of a 27-year-old immunocompetent man who progressively developed a generalized lymphadenopathy and B symptoms. Results of Epstein–Barr virus (EBV) serology were suggestive for a past infection, but the EBV viral load in whole blood was high. Also, core needle biopsy of the largest lymph node showed an image which could fit an EBV-driven reactive lymphoproliferation. Despite the absence of an immune disorder, all medical evidence points to an EBV-driven lymphoproliferative proces. In immunocompetent patients, it seems extremely uncommon to detect a high EBV viral load in the absence of serological evidence of an acute EBV infection or reactivation. We reviewed literature on this topic and on the selection of the appropriate sample type for EBV PCR, as this is known to be a critical point. Serological testing for the diagnosis of EBV infection is the gold standard in immunocompetent patients. Measuring EBV viral load is only recommended when dealing with immunocompromised patients. Although extremely rare, this case report shows that there is a place for EBV PCR in certain situations in immunocompetent patients. Besides, there is still no consensus regarding the specimen of choice for the determination of the EBV viral load. The preferred specimen type seems to depend on the patient’s underlying condition.

Epstein-Barr virus viral load and serology in childhood non-Hodgkin's lymphoma and chronic inflammatory conditions in Uganda: implications for disease risk and characteristics.

Epstein-Barr virus (EBV) has been linked to malignancies and chronic inflammatory conditions. In this study, EBV detection was compared in children with non-Hodgkin's lymphoma and children with chronic inflammatory conditions, using samples and data from a case-control study carried out at the Mulago National Referral Hospital between 2004 and 2008. EBV viral load was measured in saliva, whole blood and white blood cells by real-time PCR. Serological values for IgG-VCA, EBNA1, and EAd-IgG were compared in non-Hodgkin's lymphoma and chronic inflammatory conditions; and in Burkitt's lymphoma and other subtypes of non-Hodgkin's lymphoma. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated. Of the 127 children included (87 males and 40 females; median age 7 years, range 2-17), 96 had non-Hodgkin's lymphoma (46 Burkitt's lymphoma and 50 other non-Hodgkin's lymphoma), 31 had chronic inflammatory conditions, and only 10% were HIV-positive. The most common clinical presentations for all disease categories considered were fever, night sweats, and weight loss. EBV viral load in whole blood was elevated in Burkitt's lymphoma compared to other non-Hodgkin's lymphoma (OR 6.67, 95% CI 1.32, 33.69; P-value = 0.04), but EBV viral loads in saliva and white blood cells were not different in any of the disease categories considered. A significant difference in EAd-IgG was observed when non-Hodgkin's lymphoma was compared with chronic inflammatory conditions (OR 0.19, 95% CI 0.07, 0.51; P-value = 0.001). When compared to chronic inflammatory conditions, EBV viral load was elevated in Burkitt's lymphoma, and EA IgG was higher in non-Hodgkin's lymphoma. This study supports an association between virological and serological markers of EBV and childhood non-Hodgkin's lymphoma, irrespective of subtype, in Uganda.

EBV Load in Whole Blood Correlates With LMP2 Gene Expression After Pediatric Heart Transplantation or Allogeneic Hematopoietic Stem Cell Transplantation

Epstein-Barr virus (EBV) is associated with posttransplant lymphoproliferative disease (PTLD), and EBV load measurement is an important tool to monitor transplant patients. Although EBV DNA quantification has high sensitivity to identify patients at risk for PTLD, it lacks specificity. We examined whether EBV gene expression in peripheral B cells can increase specificity or correlates with EBV load. Altogether, 220 blood samples were collected from pediatric patients after heart transplantation (HTx, n=57), renal transplantation (n=1), or hematopoietic stem cell transplantation (n=21). In each blood sample, EBV load was quantified in whole blood, plasma, and B cells using qPCR. Additionally EBV gene expression (EBNA2, LMP1, LMP2, and BZLF1) in B cells was analyzed using relative quantitative RT-qPCR. Positive expression of at least one gene was detected in 112 (51%) of 220 samples. Patients with PTLD or chronic high viral loads after solid organ transplantation exhibited no homogeneous EBV gene expression pattern. Expression of LMP2, LMP1, or EBNA2 was only observed when EBV load exceeded 1000 copies/mL. A high correlation between the level of LMP2 expression and EBV load in B cells or whole blood was observed (ρ=0.72 or ρ=0.6, HTx population). The analysis of EBV gene expression in peripheral B cells does not provide additional information about patients' risk of developing PTLD. As EBV load in whole blood correlates well with LMP2 gene expression in EBV-infected B cells, EBV DNA quantification in whole blood alone seems to be a sufficient tool to monitor these patients.

Comparison of EBV DNA viral load in whole blood, plasma, B‐cells and B‐cell culture supernatant

Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios.

Reduced IL-7 Responsiveness Defined by Signal Transducer and Activator of Transcription 5 Phosphorylation in T Cells May Be a Marker for Increased Risk of Developing Cytomegalovirus Disease in Patients after Hematopoietic Stem Cell Transplantation

Cytomegalovirus (CMV) reactivation may lead to CMV disease associated with high morbidity and mortality in patients after hematopoietic stem cell transplantation (HSCT); the identification of clinically relevant markers may aid in the identification of patients at increased risk for developing CMV-associated complications. We evaluated the phosphorylation of signal transducer and activator of transcription 5 (STAT5) in CD4+ T cells, CD8+ T cells, and TCRγδ T cells in response to stimulation with IL-7 or IL-2 after HSCT by analyzing blood samples taken monthly 1 to 6 months after HSCT. Patients were monitored weekly with a quantitative PCR from the time of engraftment for CMV viral load in whole blood until at least day 100 after HSCT. We identified a correlation between clinical outcome regarding CMV replication and the ability to respond to IL-7 and IL-2 defined by STAT5 phosphorylation (pSTAT5). Patients with recurrent or prolonged CMV replications had significantly lower pSTAT5 upon stimulation of T cells with either IL-7 or IL-2 at time points 1 through 3 than those without CMV replication (P < .05). This was also found after stimulation of CD8+ T cells at time point 2 (P < .05). We conclude that reduced responses to IL-7, reflected by pSTAT5, may represent a clinically relevant functional biomarker for individuals at increased risk for CMV reactivation; our data may also aid in designing better strategies to improve anti-CMV immune responses without increasing the risk of developing graft-versus-host disease.

Cytomegalovirus load in whole blood is more reliable for predicting and assessing CMV disease than pp65 antigenaemia

CMV is a common cause of disease in immunocompromised patients. Because sampling of the diseased organ can be invasive, markers of systemic CMV reactivation such as pp65 and CMV viral load are commonly used to monitor patients at risk of CMV disease. In this retrospective analysis, the performance of these markers was compared in solid organ transplant recipients, patients with haematological malignancies and HIV infection. Both assays were sensitive markers of reactivation, however, the predictive value for disease of a positive result for both was low. Compared to viral load, the pp65 assay was a less sensitive marker of CMV reactivation. It was only positive when the viral load was greater than 3 log (10) copies/ml whole blood and was negative in 10 instances when the viral load was between 3 and 5 logs.In concordantly positive samples, the number of pp65 positive cells varied widely relative to the viral load and the number of positive cells counted could not be used to predict disease likelihood with any certainty.To conclude, CMV viral load provides a more consistent guide to determine likelihood of disease than pp65 count and is a more sensitive marker of CMV reactivation.

Reactivation of intestinal CMV in a renal transplant patient after 10 years from the transplant

Introduction.We analyzed the clinical case of a 51 years old man, kidney transplanted on December 2002. On April 2011, he had acute rectal bleeding, renal chronic rejection (creatinine 2.9 mg/dl), Hgb 8.7 g/dl, positive anti-CMV antibodies (IgG). A colonoscopy showed diverticulosis of the rectum associated with deepithelialisation. The patient was treated with maintenance immunosuppressive post-transplant therapy. On June 2011, the colonoscopy showed a stenosing lesion of the sigmoid colon, and blood sampling and intestinal biopsy were performed to search Cytomegalovirus (CMV) DNA by PCR. Methods. The presence of CMV-DNA was sought by automatic extractor QIACUBE, using QIAamp DNA BLOOD Mini Kit (Qiagen) for whole blood and QIAamp DNA Mini Kit (Qiagen) for biopsy.The extracted DNA was then amplified by Real Time PCR using Q-CMV RealTime Complete Kit (Nanogen), on instrument Applied Biosystems 7300. Results. At disease onset the viral load in whole blood was 208000 Geq/ml, and biopsy was positive. Antiviral therapy with Ganciclovir led to the negativity of the viral load and remission of symptoms. Conclusions. The clinical case described presented a reactivation of CMV infection in the intestine after more than 10 years from kidney transplantation, while the highest incidence of CMV reactivation usually occurs during the first year. In our opinion, the reactivation can be traced to long-term immunosuppressive therapy (maintenance posttransplant therapy) in combination with a state of inflammation of the intestinal mucosa. In fact, patients with IBD treated with steroid drugs, in particular the group of refractory to therapy and thus have a recovery of the inflammatory process, are exposed to reactivation of CMV with intestinal localization.

Comparative evaluation of the QIAsymphony RGQ system with the easyMAG/R-gene combination for the quantitation of cytomegalovirus DNA load in whole blood

BackgroundThe detection of cytomegalovirus (CMV) DNA in blood is a key feature of the virological surveillance of immunocompromised patients.MethodsThe QIAsymphony RGQ system (QIAGEN S.A.S., France) combines the extraction/distribution steps on QIAsymphony SP/AS instruments with amplification on a Rotor-Gene Q RT-PCR machine. This system was compared to a strategy combining an extraction step on the NUCLISENS easyMAG platform (bioMérieux) with the CMV R-gene kit (Argene) on 100 whole blood specimens collected from immunocompromised patients of the University Hospital of Saint-Etienne, France.ResultsThe overall agreement between the two strategies was 86% (kappa coefficient of 0.67); the 14 discrepant results corresponded to low DNA loads. The 62 samples found positive with both tests were correlated (Pearson r coefficient of 0.70, P < 0.01) despite an over quantitation of 0.25 log10 copies/ml with the easyMAG/Argene strategy (P < 0.001). Very close results were also obtained with a commercial panel of 10 samples with CMV loads ranging from 2.36 to 6.41 log10 copies/ml. The inter-run and intra-run variability was consistently lower with the QIAGEN platform.ConclusionsThese results validate the performance of the QIAsymphony RGQ system for the routine quantitation of CMV DNA. This fully-automated platform reduces the hands-on time and improves standardization, traceability and quality control assessment.

Distribution of hepatitis C virus in circulating blood components from blood donors

Current nucleic acid tests (NAT) for blood donor screening use plasma as the test sample and, consequently, cannot detect virions bound to blood cells of infected donors. Hepatitis C virus (HCV) RNA and infectious virions have been detected in association with the cellular components of blood of patients with active liver disease; however, studies comparing HCV viral loads in whole blood and plasma have generated contradictory results. The aim of this study was to investigate the distribution of HCV in different compartments of the peripheral blood from HCV-infected blood donors, which may differ from that observed in patients with HCV-associated liver disease. Hepatitis C virus-positive donor specimens were identified by NAT and antibody testing. HCV RNA was extracted from samples of whole blood and their corresponding components (RBC and plasma). Viral RNA was quantified by real-time qRT-PCR. Hepatitis C virus was present in all blood components from infected donors from which RNA could be amplified. For the majority of samples, plasma (34/46) had the highest detectable concentration of HCV RNA, and RBC (37/46) had the lowest. Specimens with negative NAT and positive antibody assays also produced qRT-PCR negative results. These results indicate that including the RBC fraction in the tested sample will not increase assay sensitivity. Although 10% of the specimens had a higher viral load in whole blood, there was no significant overall increase in sensitivity to justify changes in the specimen format. Thus, plasma specimens are well suited for blood donor screening for HCV.

Comparison of Commercial Extraction Systems and PCR Assays for Quantification of Epstein-Barr Virus DNA Load in Whole Blood

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.

Comparison of QIAsymphony Automated and QIAamp Manual DNA Extraction Systems for Measuring Epstein-Barr Virus DNA Load in Whole Blood Using Real-Time PCR

Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively). For samples estimated as having ≥10,000 copies/mL, the intrarun and interrun variations were significantly lower using QIAsymphony (10.0% and 6.8%, respectively), compared with QIAamp (18.6% and 15.2%, respectively); for samples having ≤1000 copies/mL, the two variations ranged from 27.9% to 43.9% and were not significantly different between the two systems. Among 68 paired clinical samples, 48 pairs yielded viral loads ≥1000 copies/mL under both extraction systems. Although the logarithmic linear correlation from these positive samples was high (r(2) = 0.957), the values obtained using QIAsymphony were on average 0.2 log copies/mL higher than those obtained using QIAamp. Thus, the QIAsymphony and QIAamp systems provide similar EBV DNA load values in whole blood.

Current and past Epstein-Barr virus infection in risk of initial CNS demyelination

To assess risk of a first clinical diagnosis of CNS demyelination (FCD) in relation to measures of Epstein-Barr virus (EBV) infection within the context of other known risk factors. This was a multicenter incident case-control study. FCD cases (n = 282) aged 18-59 years and controls (n = 558, matched on age, sex, and region) were recruited from 4 Australian centers between November 1, 2003, and December 31, 2006. A nested study (n = 215 cases, n = 216 controls) included measurement of whole blood quantitative EBV DNA load and serum EBV-specific antibodies. Conditional logistic regression was used to analyze case-control differences. There were no significant case-control differences in the proportion with detectable EBV DNA (55.8% vs 50.5%, respectively, p = 0.28), or in quantitative EBV DNA load (p = 0.33). Consistent with previous work, higher anti-EBV-specific immunoglobulin G (IgG) titers and a history of infectious mononucleosis were associated with increased FCD risk and there was an additive interaction with HLA-DRB1*1501 status. We found additional interactions between high anti-EBNA IgG titer and SNPs in HLA-A (adjusted odds ratios [AOR] = 19.84 [95% confidence interval (CI) 5.95 to 66.21] for both factors compared to neither) and CTLA-4 genes (AOR = 0.31 [95% CI 0.13 to 0.76] for neither factor compared to both). EBV DNA load was lower at higher serum 25-hydroxyvitamin D concentrations in controls (r = -0.17, p = 0.01). An adverse effect of higher EBV DNA load on FCD risk was increased with higher 25-hydroxyvitamin D concentration (p[interaction] = 0.02). Past infection with EBV, but not current EBV DNA load in whole blood, is significantly associated with increased FCD risk. These associations appear to be modified by immune-related gene variants.

Epstein-Barr virus DNA load in peripheral blood mononuclear cells and whole blood from pediatric transplant recipients

Monitoring of circulating Epstein-Barr virus (EBV) DNA in pediatric transplant patients has been shown to be useful in post-transplant patient management. It still remains unclear which blood sample type is more suitable, and how EBV DNA levels in whole blood (WB) correlate with those in peripheral blood mononuclear cells (PBMCs). The aim of this study was to compare EBV DNA load in WB and PBMCs of pediatric transplant recipients. After liver, kidney, or combined liver-kidney transplantation, 172 matched WB and PBMCs samples were collected from 84 children (130 samples from 42 patients consisted of multiple collections). The EBV DNA level in PBMCs was determined by home-made real-time polymerase chain reaction using TaqMan chemistry. In parallel, the viral load (VL) in WB was measured by a commercial LightCycler EBV Quant Kit. The EBV DNA levels and dynamics of VL changes were assessed and compared between WB and PBMCs. The overall correlation between EBV DNA level in PBMCs and WB was statistically significant and high, r(2) =0.87 (P<0.001). However, the sensitivity of EBV detection was lower in WB (93.9%). Longitudinal analysis of EBV DNA load dynamics in PBMCs and WB indicated that EBV DNA load fluctuations were larger in WB, but the trend of decreases and increases, with minor exceptions, was similar in both sample types. The high correlation of EBV DNA levels, as well as the similar dynamics of EBV DNA changes in both sample types, make WB a good alternative to EBV DNA monitoring in PBMCs of pediatric transplant recipients. However, the subtle increase of the VL may be detected earlier in PBMCs.

The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation

Background Monitoring of EBV DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary, but not sufficient, to identify patients at risk of EBV-induced post-transplantation lymphoproliferative disorders (PTLD). Combining this with quantifying EBV-specific cellular immunity was shown to be helpful. In this study, we evaluated the value of IFNγ-Elispot assay in monitoring EBV DNAemia after HSCT. Methods EBV-DNA load in whole blood was monitored at least weekly using real-time PCR in 40 recipients of HSCT. Quantitative and qualitative T-cell recoveries, including EBV-specific T-cell quantification by Elispot assay, were studied 60, 100, 180 and 360 days after HSCT. Results Among the 35 evaluable patients, 14 (35%) presented EBV DNAemia, only 2/14 (14%) needing pre-emptive treatment with rituximab. The greatest risk factor for EBV DNAemia was the presence of anti-thymocyte globulin (ATG) (p = 0.005). EBV-specific cellular immune recovery was monitored by IFNγ-Elispot assay. Using multivariate analysis , four factors were found to significantly influence IFNγ-Elispot results at defined times post-HSCT: EBV DNAemia, young age, global T-cell recovery and severe acute GVHD. In those cases where EBV DNAemia occurred and cleared spontaneously, Elispot results gave more than 1000 spot-forming cells (SFC)/10 6 PBMC. Conclusion Elispot assay may be usefully combined with EBV-DNA load monitoring to determine when a patient should receive pre-emptive treatment, or when the clinician should avoid Rituximab use which severely immunocompromises patients.

Infections aiguës à herpèsvirus humain 6 (HHV-6) : quand et comment traiter ?

The pathogenicity of human herpesvirus 6 (HHV-6) still raises numerous questions. Acute HHV-6 infections correspond to primary infections, reactivations or exogenous reinfections. The expression of related clinical symptoms is highly variable but may be extremely severe, particularly among immunocompromised patients. The prototypic severe disease associated with these infections is limbic encephalitis occurring in stem cell transplant recipients. The diagnosis of acute HHV-6 infections relies on the quantitation of viral load in whole blood by means of quantitative PCR. The demonstration of HHV-6 causative role in the genesis of clinical symptoms requires additional investigations such as the search for HHV-6 DNA in cerebrospinal fluid in case of encephalitis. The chromosomal integration of HHV-6 DNA is a rare event among HHV-6-infected subjects but may alter the interpretation of virological results. Therapy with ganciclovir, foscarnet or cidofovir has not yet clear indications but, at the current stage of knowledge, only concerns the treatment of highly symptomatic infections. The usefulness of prophylactic or pre-emptive antiviral chemotherapy has not yet been convincingly demonstrated. Treatment efficacy must be checked through a clinical and virological follow up, based in part on quantitative PCR approaches. Controlled studies are urgently needed with the goal of evaluating the cost-effectiveness of HHV-6 follow up and therapy in different clinical situations.

PX-24 Comparative studies of the Access® CMV IgG assay

Reliable real-time quantitative PCR assays to measure Epstein-Barr virus (EBV) DNA load (EBV) are useful for monitoring EBV-associated diseases. We evaluated a new commercial kit, EBV R-gene Quantification kit (Argene, Varilhes, France) to quantify EBV DNA load in whole blood. Assay performance was assessed with two PCR platforms (LightCycler 2.0 and SmartCycler 2.0) and three commercial DNA extraction methods. The assay was compared with our in-house real-time EBV PCR using samples from the Quality Control for Molecular Diagnostics 2006 EBV proficiency program and using 167 whole-blood specimens from individuals with infectious mononucleosis, from transplanted or HIV-infected patients, and from EBV-seropositive healthy carriers. The EBV R-gene assay was sensitive to 500 copies of EBV DNA per milliliter of whole blood with the two PCR platforms and the three extraction methods and was linear across 4 orders of magnitude. Intra- and interassay coefficients of variations were less than 20%. Nine of 10 samples tested with the EBV R-gene were in agreement with the expected qualitative results of the Quality Control for Molecular Diagnostics 2006 EBV proficiency program, and 7 of 10 samples were within ±0.5 log units of the expected quantitative values, with discrepant results mostly observed for low viral load (ie, <1000 copies/ml). In the clinical specimens, the correlation between the R-gene assay and the in-house PCR was high (r = 0.92). In conclusion, the EBV R-gene assay accurately assesses the EBV DNA load in whole blood of patients with various forms of EBV infections.

Different expression of human herpesvirus-6 (HHV-6) load in whole blood may have a significant impact on the diagnosis of active infection

Background Viral load in whole blood is the main virological marker for assessing HHV-6 infection and is used as an indication to begin antiviral therapy. Results are usually expressed as the number of genomic equivalent copies (gec) per mL of blood, although HHV-6 DNA in blood is mainly localized in lymphocytes and polymorphonuclear leukocytes. Objectives Since leukocyte counts vary in immunocompromised patients, especially in stem cell transplant recipients, the aim of this study was to compare HHV-6 load expressed as gec per mL with load expressed as gec per million cells (mc), using quantitative real-time PCR for HHV-6 and cell DNA. Study design 194 blood samples from 101 patients were analyzed. Leukocyte count was obtained for 142 samples. Results The two modes of expression were incompletely correlated ( p < 0.0001; R 2 = 0.732). To understand this relative discrepancy, samples were classified according to hematological criteria (normal leukocyte count, leukopenia, agranulocytosis, lymphopenia). The expression modes were correlated in all cases except for agranulocytosis ( p = 0.21; R 2 = 0.087). Moreover, the median of ratio between gec per mc and gec per mL ranged from 0.5 when leukocyte count was normal, to 8.2 in cases of agranulocytosis. HHV-6 load follow-up suggested that in agranulocytosis expressing results as gec per mc tended to provide a more representative result. Conclusions The different expression of HHV-6 load in whole blood, as either gec per mL or gec per mc resulted in different estimations of infection in the case of agranulocytosis. In this situation, the latter mode of expression is preferred.

Epstein-Barr Virus Load in Whole Blood Correlates With HIV Surrogate Markers and Lymphoma: A French National Cross-Sectional Study

Epstein-Barr Virus Load in Whole Blood Correlates With HIV Surrogate Markers and Lymphoma: A French National Cross-Sectional Study

Contribution of human herpesvirus 6 (HHV-6) viral load in whole blood and serum to investigate integrated HHV-6 transmission after haematopoietic stem cell transplantation

Background Human herpesvirus 6 (HHV-6) is susceptible to latency and recurrence. A less-frequent form of HHV-6 persistence is the integration of viral DNA into host chromosomes. Objectives To investigate HHV-6 viral load after haematopoietic stem cell transplantation (HSCT) in whole blood (WB) and serum with regard to integrated HHV-6 transmission diagnosis. Study design HHV-6 DNA quantitation in serum and WB was performed using quantitative polymerase chain reaction for the follow-up of a 16-year-old girl after HSCT. In whole blood, results were expressed as HHV-6 genomic equivalent copies (gec) per milliliter of WB or per million cells. Results HHV-6 viral load (undetectable before HSCT) increased up to 3.05 × 10 7 gec/10 6 cells. HHV-6 viral load in the donor sample (3.44 × 10 6 gec/10 6 cells) was in favor of viral transmission through HSCT. The correlation between viral load in WB and serum was significant ( p = 0.0005). Viral load results expressed as gec/10 6 cells in WB was more reliable than results expressed as gec/ml of whole blood. Conclusion These findings indicate that HHV-6 may be transmitted during HSCT as integrated virus contained in the graft. This reiterates that in the setting of HSCT, HHV-6 viral load must be correctly interpreted. Using HHV-6 viral load expressed as gec/10 6 cells may be more suitable for the follow-up of patients with integrated HHV-6.

Epstein-Barr virus load in whole blood is associated with immunosuppression, but not with post-transplant lymphoproliferative disease in stable adult heart transplant patients

Development of Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is a serious complication following heart transplantation (HTX). This study investigates EBV DNA load in adult heart transplant recipients, its association with immunosuppression, and its potential as a marker for development of PTLD. EBV DNA load was measured prospectively by quantitative real-time polymerase chain reaction (PCR) in 172 stable HTX patients. Sixty-seven patients (39.0% of total) had a positive EBV PCR at initial examination [median 4.9 (range 1.1-16.9) years post-HTX]. In follow-up testing of 67 positive patients 6 months later, 36 patients continued to have a positive EBV PCR. Overall incidence of EBV DNA was significantly associated with calcineurin inihibitors, azathioprine medication, and with the absence of mycophenolate mofetil (MMF) treatment. In patients with positive EBV DNA levels at initial examination and negative levels at retesting, cyclosporine A levels were found to be significantly higher at initial examination (148.4 +/- 70.2 vs. 119.6 +/- 53.5 ng/ml, P < 0.05). Three patients (1.7%, 3/172) were diagnosed with PTLD during the course of the study (mean follow up 4.0 years). EBV DNA viral load determination does not appear to be useful for risk prediction or early diagnosis of PTLD in adults after HTX, but an association of EBV DNA load with qualitative and quantitative immunosuppression is demonstrated.