Viral Load Threshold Research Articles

Determination of a Viral Load Threshold To Distinguish Symptomatic versus Asymptomatic Rotavirus Infection in a High-Disease-Burden African Population.

We evaluated quantitative real-time PCR to establish the diagnosis of rotavirus gastroenteritis in a high-disease-burden population in Malawi using enzyme immunoassay as the gold standard diagnostic test. In 146 children with acute gastroenteritis and 65 asymptomatic children, we defined a cutoff point in the threshold cycle value (26.7) that predicts rotavirus-attributable gastroenteritis in this population. These data will inform the evaluation of direct and indirect rotavirus vaccine effects in Africa.

Merkel cell carcinoma: histopathologic and prognostic features according to the immunohistochemical expression of Merkel cell polyomavirus large T antigen correlated with viral load.

Merkel cell carcinoma (MCC) is a neuroendocrine skin malignancy frequently associated with Merkel cell polyomavirus (MCPyV), which is suspected to be oncogenic. In a series of MCC patients, we compared clinical, histopathologic, and prognostic features according to the expression of viral large T antigen (LTA) correlated with viral load. We evaluated the LTA expression by immunohistochemistry using CM2B4 antibody and quantified viral load by real-time polymerase chain reaction. We analyzed formalin-fixed, paraffin-embedded (FFPE) tissue samples (n = 36) and corresponding fresh-frozen biopsies when available (n = 12), of the primary tumor and/or metastasis from 24 patients. MCPyV was detected in 88% and 58% of MCC patients by real-time polymerase chain reaction and immunohistochemistry, respectively. The relevance of viral load measurements was demonstrated by the strong consistency of viral load level between FFPE and corresponding frozen tissues as well as between primary tumor and metastases. From FFPE samples, 2 MCC subgroups were distinguished based on a viral load threshold defined by the positivity of CM2B4 immunostaining. In the LTA-negative subgroup with no or low viral load (nonsignificant), tumor cells showed more anisokaryosis (P = .01), and a solar elastosis around the tumor was more frequently observed (P = .03). LTA-positive MCCs with significant viral load had a lower proliferation index (P = .03) and a longer survival of corresponding patients (P = .008). Depending on MCPyV involvement, 2 MCC subgroups can be distinguished on histopathologic criteria, and the CM2B4 antibody is able to differentiate them reliably. Furthermore, the presence of a significant viral load in tumors is predictive of better prognosis.

1565Identifying Optimal HIV Viral Load (VL) Thresholds for Predicting Antiretroviral Treatment Failure (TF) Using ROC Curve Analysis

Background: Guidelines on HIV treatment differ globally in recommended viral load (VL) thresholds indicative of treatment failure (TF). Improvements in VL assay sensitivity and worldwide scale-up of VL monitoring make it increasingly important to determine optimal VLs thresholds for guiding treatment changes. Receiver operating characteristic (ROC) curves can identify optimal VL thresholds which maximize sensitivity and specifi city for predicting TF. The BRAVO study was a retrospective study, using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0, to explore thresholds of VL that might predict subsequent TF. Methods: Longitudinal patient specimens were collected through the VERxVE study, a randomized, double-blind (DB) study in treatment-naive patients treated with nevirapine immediate-release or extended-release (VIRAMUNE IR or XR), plus emtricitabine and tenofovir DF. Stored plasma samples from up to 24 timepoints (pre-treatment through 144 weeks of DB treatment), were tested with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0. The BRAVO patient endpoints of TF (which differed from the primary VERxVE endpoints) were determined by two independent HIV physicians based on VL trajectories. Guideline-based defi nitions of TF were also examined. A simple logistic regression model and ROC curves were created to determine the optimal VL thresholds for predicting TF at key study timepoints. Results: Of 563 evaluable patient-series, 74 patients (13%) had TF. By ROC curve analysis, a VL threshold of 95 copies/ml at week 24 and 33 copies/ml at week 48 maximized sensitivity and specifi city of predicting eventual TF [Area Under Curve: 0.75 and 0.73, respectively]. Overall sensitivity and specifi city were similar when compared to guidelines-based VL thresholds of 50 or 200 copies/ml and when using thresholds from an endpoint-based TF defi nition. Conclusions: ROC curve analysis identifi ed a VL threshold that maximized sensitivity and specifi city in predicting TF. Viral load thresholds of 50 or 200 copies/ml can also be used without a signifi cant decrease in overall sensitivity and specifi city. As VL assays may differ in sensitivity, ROC curve analysis can help optimize the clinical utility of VL monitoring.

Herpes simplex virus-2 transmission probability estimates based on quantity of viral shedding

Herpes simplex virus (HSV)-2 is periodically shed in the human genital tract, most often asymptomatically, and most sexual transmissions occur during asymptomatic shedding. It would be helpful to identify a genital viral load threshold necessary for transmission, as clinical interventions that maintain viral quantity below this level would be of high utility. However, because viral expansion, decay and re-expansion kinetics are extremely rapid during shedding episodes, it is impossible to directly measure genital viral load at the time of sexual activity. We developed a mathematical model based on reproducing shedding patterns in transmitting partners, and median number of sex acts prior to transmission in discordant couples, to estimate infectivity of single viral particles in the negative partner's genital tract. We then inferred probability estimates for transmission at different levels of genital tract viral load in the transmitting partner. We predict that transmission is unlikely at viral loads less than 10(4) HSV DNA copies. Moreover, most transmissions occur during prolonged episodes with high viral copy numbers. Many shedding episodes that result in transmission do not reach the threshold of clinical detection, because the ulcer remains very small, highlighting one reason why HSV-2 spreads so effectively within populations.

Clinical Utility of Viral Load in Management of Cytomegalovirus Infection after Solid Organ Transplantation

The negative impact of cytomegalovirus (CMV) infection on transplant outcomes warrants efforts toward improving its prevention, diagnosis, and treatment. During the last 2 decades, significant breakthroughs in diagnostic virology have facilitated remarkable improvements in CMV disease management. During this period, CMV nucleic acid amplification testing (NAT) evolved to become one of the most commonly performed tests in clinical virology laboratories. NAT provides a means for rapid and sensitive diagnosis of CMV infection in transplant recipients. Viral quantification also introduced several principles of CMV disease management. Specifically, viral load has been utilized (i) for prognostication of CMV disease, (ii) to guide preemptive therapy, (iii) to assess the efficacy of antiviral treatment, (iv) to guide the duration of treatment, and (v) to indicate the risk of clinical relapse or antiviral drug resistance. However, there remain important limitations that require further optimization, including the interassay variability in viral load reporting, which has limited the generation of standardized viral load thresholds for various clinical indications. The recent introduction of an international reference standard should advance the major goal of uniform viral load reporting and interpretation. However, it has also become apparent that other aspects of NAT should be standardized, including sample selection, nucleic acid extraction, amplification, detection, and calibration, among others. This review article synthesizes the vast amount of information on CMV NAT and provides a timely review of the clinical utility of viral load testing in the management of CMV in solid organ transplant recipients. Current limitations are highlighted, and avenues for further research are suggested to optimize the clinical application of NAT in the management of CMV after transplantation.

Evaluation of the False Recent Classification Rates of Multiassay Algorithms in Estimating HIV Type 1 Subtype C Incidence

Laboratory cross-sectional assays are useful for the estimation of HIV incidence, but are known to misclassify individuals with long-standing infection as recently infected. The false recent rate (FRR) varies widely across geographic areas; therefore, accurate estimates of HIV incidence require a locally defined FRR. We determined FRR for Botswana, where HIV-1 subtype C infection is predominant, using the BED capture enzyme immunoassay (BED), a Bio-Rad Avidity Index (BAI) assay (a modification of the Bio-Rad HIV1/2+O EIA), and two multiassay algorithms (MAA) that included clinical data. To estimate FRR, stored blood samples from 512 antiretroviral (ARV)-naive HIV-1 subtype C-infected individuals from a prospective cohort in Botswana were tested at 18-24 months postenrollment. The following FRR mean (95% CI) values were obtained: BED 6.05% (4.15-8.48), BAI 5.57% (3.70-8.0), BED-BAI 2.25% (1.13-4.0), and a combination of BED-BAI with CD4 (>200) and viral load (>400) threshold 1.43% (0.58-2.93). The interassay agreement between BED and BAI was 92.8% (95% CI, 90.1-94.5) for recent/long-term classification. Misclassification was associated with viral suppression for BED [adjusted OR (aOR) 10.31; p=0.008], BAI [aOR 9.72; p=0.019], and MAA1 [aOR 16.6; p=0.006]. Employing MAA can reduce FRR to <2%. A local FRR can improve cross-sectional HIV incidence estimates.

Human cytomegalovirus detection by real-time PCR and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries

Aim: Cytomegalovirus (CMV) infection is one of the most common complications in patients submitted to hematopoietic stem cell transplant (HSCT). Pre-emptive therapy has been indicated in patients with laboratory evidence of CMV replication. The aims of this study were to compare real-time PCR or pp65 antigen assay methodologies to detect CMV replication in HSCT patients, define a viral load threshold for initiation of pre-emptive therapy, and assess the feasibility of its implementation in hospitals of countries with low and middle income.Material and methods: Human CMV detection by real-time PCR and pp65 antigen assay was carried out in blood and plasma samples of HSCT patients collected weekly during 3 months. Pre-emptive therapy was based on CMV antigenemia results.Results: Twenty-one patients were monitored with a total of 227 samples collected; 13 (62%) patients were children. A poor correlation was observed between qualitative results, though quantitative results showed statistically significant difference, with higher viral loads detected in patients with positive antigenemia. Compared to a positive antigenemia, a cutoff value of 1067·5 copies/ml, 3·03 log10/ml, for viral load was obtained with 100% sensitivity and 71% specificity.Conclusion: CMV real-time PCR in whole blood was suitable for monitoring HSCT patients. However, its high cost is a limiting factor, and it could be used to monitor selected patients, those with prolonged leukopenia and underweight children, and subsequently switched to pp65 antigen test. Further studies involving larger numbers of patients should be performed to confirm this statement.

Validation of an algorithm to identify antiretroviral-naïve status at time of entry into a large, observational cohort of HIV-infected patients

Large, observational HIV cohorts play an important role in answering questions which are difficult to study in randomized trials; however, they often lack detailed information regarding previous antiretroviral treatment (ART). Knowledge of ART treatment history is important when ascertaining the long-term impact of medications, co-morbidities, or adverse reactions on HIV outcomes. We performed a retrospective study to validate a prediction algorithm for identifying ART-naïve patients using the Veterans Aging Cohort Study's Virtual Cohort-an observational cohort of 40 594 HIV-infected veterans nationwide. Medical records for 3070 HIV-infected patients were reviewed to determine history of combination ART treatment. An algorithm using Virtual Cohort laboratory data was used to predict ART treatment status and compared to medical record review. Among 3070 patients' medical records reviewed, 1223 were eligible for analysis. Of these, 990 (81%) were ART naïve at cohort entry based on medical record review. The prediction algorithm's sensitivity was 86%, specificity 47%, positive predictive value (PPV) 87%, and negative predictive value 45%, using a viral load threshold of <400 copies/ml. Sensitivity analysis revealed that PPV would be maximized by increasing the viral load threshold, whereas sensitivity would be maximized by lowering the viral load threshold. A prediction algorithm using available laboratory data can be used to accurately identify ART-naïve patients in large, observational HIV cohorts. Use of this algorithm will allow investigators to accurately limit analyses to ART-naïve patients when studying the contribution of ART to outcomes and adverse events.

Viral load monitoring for antiretroviral therapy in resource-poor settings

Viral load monitoring for antiretroviral therapy in resource-poor settings

National Guidelines for the prevention of mother-to-child transmission of HIV across Europe - how do countries differ?

The aim was to summarize national prevention of mother-to-child transmission (PMTCT) guidelines across Europe and to identify differences between these. A survey was conducted using a structured questionnaire sent to experts in 25 European countries from January to March 2012, requesting a copy of the national guidelines. Responses were received from 23 countries. Twenty-two (96%) countries supported a policy to recommend antenatal HIV screening for all pregnant women (15: opt-out strategy; 8: opt-in strategy). For HIV-positive women in whom the only indication for antiretroviral therapy (ART) was PMTCT, the recommended gestational age for commencing ART varied from 12 to 28 weeks: initiation before 19 weeks gestation was recommended in guidelines from nine countries; in France, the UK and the Netherlands, there was a wide range, from 14 to 24 weeks, whereas the Swiss and Ukrainian guidelines recommended starting at 24-28 weeks and the German/Austrian and Lithuanian at 28 weeks. Six national guidelines recommended inclusion of Zidovudine in antenatal ART regimens, and seven (37%) allowed continuation of Efavirenz for women conceiving on this drug. According to nine guidelines, zidovudine should always be used intrapartum. Eighteen national guidelines stated that HIV-positive women on successful ART can have a vaginal delivery. Viral load thresholds for vaginal delivery were <1000 copies/ml in 5 countries, <400 copies/ml in 3 and <50 copies/ml in 11 countries. There are important differences across Europe in national PMTCT guidelines, with most variation seen where the evidence-base remains limited. Such differences should be considered when interpreting research and surveillance findings.

Simulation of Pooled Nucleic Acid Testing to Identify Antiretroviral Treatment Failure During HIV Infection in Seoul, South Korea

Simulation of Pooled Nucleic Acid Testing to Identify Antiretroviral Treatment Failure During HIV Infection in Seoul, South Korea

CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients

BackgroundSensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice. Objectives/study designUsing 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS® AmpliPrep/COBAS® TaqMan® CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS® AMPLICOR MONITOR CMV tests. ResultsThe median time between transplantation and testing samples was 57 days (range, 0–207 days). The median CMV load (log10) was 3.17IU/mL (3.21copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (−0.15 [95% CI, −0.18 to −0.13] copies/mL), but slope differences indicated only limited co-linearity. ConclusionsThe CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.

An International Multicenter Performance Analysis of Cytomegalovirus Load Tests

Background. Quantification of cytomegalovirus (CMV) load is central to the management of CMV infections in immunocompromised patients, but quantitative results currently differ significantly across methods and laboratories. Methods. The COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV test), developed using the first World Health Organization CMV standard in the calibration process, was compared to local assays used by 5 laboratories at transplant centers in the United States and Europe. Blinded plasma panels (n = 90) spiked with 2.18–6.7 log10 copies/mL and clinical plasma samples from immunocompromised patients (n = 660) were tested. Results. Observed mean panel member concentrations by site and 95% confidence intervals (CIs) of the data combined across sites were narrower for CAP/CTM CMV test compared with local assays. The 95% CI in log10 copies/mL of the combined data per panel member for CAP/CTM CMV test vs comparator assays was .17 vs 1.5 at 2.18 log10 copies/mL; .14 vs .52 at 2.74 log10 copies/mL; .16 vs .6 at 3.3 log10 copies/mL; .2 vs 1.11 at 4.3 log10 copies/mL; .21 vs 1.13 at 4.7 log10 copies/mL; and .18 vs 1.4 at 6.7 log10 copies/mL. In clinical specimens, constant and variable quantification differences between the CAP/CTM CMV test and comparator assays were observed. Conclusions. High interlaboratory agreement and precision of CAP/CTM CMV test results across 5 different laboratories over 4 orders of magnitude suggest that this assay could be valuable in prospective studies identifying clinical viral load thresholds for CMV treatment.

May viral (antigen) load be the real crucial tool leading to anergy in a “micro-evolutionary” model of host/virus interaction?

HypothesisThe hypothesis in the present work is that in host/virus/tumor (antigens) interactions, leading to anergy of the immune system, the Viral (antigen) load plays a crucial and central role, which all interactions turn around. BackgroundNotwithstanding apparent strong favorable evidences, the still prevailing concept of “active virus strategies to escape” may be misleading, since it might hide the cited pivotal role in a wide number of researches. This concept could be easily substituted by a microevolutionary model explaining many unresolved questions and allowing to emerge the role of antigen load conditioned reactions of the host’s immune system as motivated choices. EvidencesAn anergy induced condition can be detected not only in HCV, but also in the course of persistent viral (e.g. HBV, HIV) and non viral parasitic infections (e.g. Leishmania and Helminths) which share the same host’s reactions leading to anergy, independently on the infecting agents. The starting point of those reactions is always time elapsing from the primary infection after a short early (often undetected) period of high viral(antigen) load in the lack of clearance. This latter seems then the only conceivable link between such so different infections determining, as far as HBV and HCV are concerned, also Hepatocarcinoma under indirect facilitating conditions.In a wide majority of studies it seems clearly evident that viral load exerts a main role which contributes to determine host chosen reactions aimed at avoiding dangerous outcomes while controlling viral load. Strong clinical (i.e. both HIV infected patients treated with HAART, and helminths infected people with deworming drugs acting directly on viral and parasitic loads) and experimental studies (i.e. chimpanzees (the only animal model of HCV infection) infected with HBV inocula of different size) are here reported or cited to highlight the crucial role of antigen load also on HIV infection transmission, seroconversion, disease progression, treatment initiation and efficacy. Concluding suggestionsThe new era for antiviral drugs like protease and polymerase inhibitors that seem to be more efficacious and less toxic than Ribavirin, may open the possibility to verify, when administered during the early phase of HCV infection (eventually helped by an immune-stimulant cytokine as IL-2), whether a precocious significant reduction of viral load (threshold) may allow the host to sustain his strong reactions and clear the virus within the due time, confirming the hypothesis about the crucial role of this tool which may be extended to all the cited infections.

Time profile of viral DNA in aqueous humor of patients treated for VZV acute retinal necrosis using quantitative real‐time PCR

AbstractPurpose To evaluate the kinetics of varicella zoster virus (VZV) load using quantitative PCR (qPCR) in patients treated for acute retinal necrosis (ARN). Design: Cohort study, evaluation of diagnostic test or technology.Methods Six patients (52±13 years) with ARN syndrome were consecutively studied. Aqueous humor (AH) was sampled from all eyes for qPCR evaluation. The patients were treated with intravenous aciclovir and intravitreal injections of antiviral drugs. The mean follow‐up was 17.6±16.4 months.Results Two main portions of the viral load curves were observed for each patient: a plateau phase (27.8±24.9 days) followed by a decrease in the number of viral genome copies. The mean baseline viral load was 3.4×107±4.45×107copies/ml (6×106 to 1.2×108). The viral load decreased following a logarithmic model, with a 50% reduction obtained in 3±0.7 days. There was a significant viral load (&gt; 102 copies/ml) at 50 days after the onset of treatment, despite antiviral drugs.Conclusion qPCR use demonstrated reproducible VZV DNA kinetics with a two‐phase evolution: plateau followed by logarithmic decrease. These data suggest that high‐dosage antiviral therapy during the conventional 10 days duration is insufficient in most patients. This patient's series responded with a similar decrease in viral load once initiated, this may be used to predict the progression of future patients. The correlation of the viral load threshold with clinical improvement needs to be more clearly defined.

Circulating Interleukin-6 Levels Correlate with Residual HIV Viraemia and Markers of Immune Dysfunction in Treatment-Controlled HIV-Infected Patients

Antiretroviral therapy (ART)-controlled HIV-infected patients have elevated levels of systemic inflammatory markers, C-reactive protein (CRP) and interleukin (IL)-6, which correlate with increased cardiovascular risk and/or mortality. Persistent low-level viral replication could be involved in this inflammatory state. We evaluated whether residual viral load (VL) correlated with the level of systemic inflammatory/immune markers in ART-controlled HIV-infected patients. We evaluated 122 antiretroviral-controlled patients with VL 1-500 copies/ml for circulating levels of high-sensitivity (hs)CRP, hsIL-6, IL-8, soluble (s)CD14 and soluble tumour necrosis factor (TNF) receptors, sTNFR1 and sTNFR2. The patients were 80.3% men, the median age was 47 years, the median CD4(+) T-cell count was 519 cells/mm(3), the median nadir CD4(+) T-cell count was 180 cells/mm(3), the median VL was 28 copies/ml and the median body mass index was 23.3 kg/m(2). The median (range) values for IL-6, CRP, IL-8, sCD14, sTNFR1 and sTNFR2 were 0.685 pg/ml (0.15-5.46), 1.8 mg/l (0.2-9.7), 10.0 pg/ml (1.6-71.1), 1,174 ng/ml (214-3,145), 1,112 pg/ml (583-5,834) and 2,412 pg/ml (1,142-7,688), respectively. IL-6 values correlated positively with HIV VL (rho=0.217, P=0.017). The VL threshold value for significantly increased IL-6 was 31 copies/ml (P=0.023). IL-6 values correlated with markers of immune dysfunction: the CD4/CD8 ratio (rho=-0.248, P=0.011), CD4 nadir level (rho=-0.186, P=0.04) and nadir CD4/CD8 ratio (rho=-0.257, P=0.008). They negatively correlated with markers of immune activation sCD14 (rho=-0.236, P=0.011) and IL-8 (rho=-0.290, P=0.002). We found no correlation between VL and CRP or other markers of inflammation/immune dysfunction including sTNFR1, sTNFR2, sCD14 and IL-8. We report here that low-range IL-6 levels correlated with low-range VL and inversely with sCD14 and IL-8. Our findings suggest that maintaining VL<30 copies/ml in HIV-infected patients might therefore reduce IL-6.

Evaluation of Viral Load Thresholds for Predicting New World Health Organization Stage 3 and 4 Events in HIV-Infected Children Receiving Highly Active Antiretroviral Therapy

This study evaluated a wide range of viral load (VL) thresholds to identify a cut-point that best predicts new clinical events in children on stable highly active antiretroviral therapy (HAART). Cox proportional hazards modeling was used to assess the adjusted risk for World Health Organization stage 3 or 4 clinical events (WHO events) as a function of time-varying CD4, VL, and hemoglobin values in a cohort study of Latin American children on HAART ≥6 months. Models were fit using different VL cut-points between 400 and 50,000 copies per milliliter, with model fit evaluated on the basis of the minimum Akaike information criterion value, a standard model fit statistic. Models were based on 67 subjects with WHO events out of 550 subjects on study. The VL cut-points of >2600 and >32,000 copies per milliliter corresponded to the lowest Akaike information criterion values and were associated with the highest hazard ratios (2.0, P = 0.015; and 2.1, P = 0.0058, respectively) for WHO events. In HIV-infected Latin American children on stable HAART, 2 distinct VL thresholds (>2600 and >32,000 copies/mL) were identified for predicting children at significantly increased risk for HIV-related clinical illness, after accounting for CD4 level, hemoglobin level, and other significant factors.

Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green–based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples

Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10(7) DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green-based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non-PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non-PMWS-affected pigs (≥ 10(2.5)). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10(7.0 ± 1.5) copies/100 ng of total DNA sample, while the cPCR detected up to 10(4.8 ± 1.5). A mean difference of 10(1.8) was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.

Utility of a commercial quantitative hepatitis C virus core antigen assay in a diagnostic laboratory setting

In this study, the utility and impact of hepatitis C virus (HCV) core antigen (Cag) detection via a commercial assay have been evaluated in diagnostic laboratory conditions. In a total of 272 samples from 226 individuals, HCV RNA was detected in 81.3% and anti-HCV antibody prevalence was 86.4%. HCV Cag reactivity was identified in 59.9% of the samples and in 75.8% with detectable RNA. The sensitivity and specificity of HCV Cag assay have been calculated as 75.8% and 95.1%, respectively, and agreement between HCV RNA and HCV Cag was moderate (κ = 0.554). HCV Cag and RNA levels were highly correlated (r = 0.915 and 0.937). A viral load threshold of 103 IU/mL has been recognized, above which the correlation with RNA became statistically significant and sensitivity increased to 90.9%. Detection and quantification of HCV core antigen have been observed as a strong alternative to nucleic acid testing for HCV monitorization.

Quantification of ostreid herpesvirus 1 (OsHV-1) in Crassostrea gigas by real-time PCR: Determination of a viral load threshold to prevent summer mortalities

French oyster farming has been experiencing severe mortalities since 2008 in Crassostrea gigas spat and juveniles mainly during the summer months. This recent phenomenon can be explained by the involvement of many factors including pathogenic agents such as the Ostreid Herpesvirus 1 (OsHV-1). From July 2008 to September 2010, 424 oyster samples received from French oyster farmers were tested to follow the concentration of OsHV-1 by quantitative real-time PCR. Comparisons of OsHV-1 loads were also made according to oyster age, ploidy, origin (hatchery-bred or wild-collected) and sample status (healthy or dying). The survey revealed that high viral loads detected in spat and juvenile oyster tissues were associated with summer mortality occurrence. No significant differences were observed in pathogen detection in relation to oyster ploidy and origin. Finally, this study allowed us to define a viral load threshold of 8.8 × 10 3 of OsHV-1 DNA per mg of tissue, which can be useful to oyster farmers in evaluating mortality risk during the different stages of oyster handling.