Performance of Two Commercially Available Automated Immunoassays for the Determination of Epstein-Barr Virus Serological Status

This study evaluated the performance of two automated Vidas (V) and Liaison (L) immunoassays for Epstein-Barr virus (EBV) serology. The detection of the viral capsid antigen (VCA) IgM, the VCA/early antigen (VCA/EA) IgG, and the Epstein-Barr nuclear antigen (EBNA) IgG was assessed on 526 sera collected for routine EBV testing in immunocompetent subjects. The determination of expected EBV status (186 EBV primary infections, 183 past EBV infections, and 157 EBV-seronegative individuals) was based on results of routine laboratory enzyme immunoassays (EIAs) together with clinical data. The sensitivity and specificity of each individual marker were determined in comparison to the expected EBV status. The agreement between the V and L profiles and the expected EBV status was established through the interpretation of combinations of the different EBV markers. Statistically significant differences between the two tests were found for the specificity of the VCA IgM marker (96.2% for V versus 93.2% for L), the sensitivity of the VCA/EA IgG marker (89% for V versus 94% for L), and the specificity of the EBNA IgG marker (96.5% for V versus 74.2% for L). The results determined for the two assays with respect to overall agreement with the established expected EBV status were not significantly different (89.7% for V versus 88.2% for L), with discrepancies mainly observed in sera referenced as primary infections. These findings demonstrated the similar performances of the Vidas and the Liaison assays for the establishment of an EBV serological status using the VCA, EA, and EBNA markers.     

Monitoring of Epstein-Barr Virus DNA Load in Peripheral Blood by Quantitative Competitive PCR

A competitive quantitative PCR (Q-PCR) assay combined with simple silica-based DNA extraction was developed for monitoring of Epstein-Barr virus (EBV) DNA load in unfractionated peripheral blood. The Q-PCR is based on competitive coamplification of a highly conserved 213-bp region of the EBNA-1 open reading frame with an internal standard (IS), added in a known concentration. The IS has the same amplicon length and base composition as the wild-type (WT) EBNA-1 amplicon but differs in 23 internally randomized bases. Competitive coamplification yields two PCR products that are quantified by enzyme immunoassay or by electrochemiluminescence detection, with probes specific for the 23 differing internal nucleotides. The Q-PCR has a sensitivity of 10 copies of either WT or IS plasmid DNA. The Q-PCR was validated by quantification of known amounts of plasmid containing the WT EBNA-1 target. Furthermore, we determined EBV genome copy numbers in different cell lines. For EBV quantification in clinical samples, DNA was isolated from lysed whole blood by silica-affinity purification. Forty-six percent of healthy donor peripheral blood samples were positive by Q-PCR. In most of these samples, viral load was less than 2,000 EBV copies/ml of blood. In peripheral blood samples from two AIDS-related non-Hodgkin's lymphoma patients, elevated EBV loads (up to 120,000 copies/ml) were observed, which decreased upon therapy. In Burkitt's lymphoma patients, up to 4,592,000 EBV genome copies/ml of blood were detected. In conclusion, the EBNA-1-based Q-PCR assay provides a reproducible, accurate, and easy method for studying the relationship between EBV load and clinical parameters.     

Cytomegalovirus and Epstein-Barr Virus DNA Kinetics in Whole Blood and Plasma of Allogeneic Hematopoietic Stem Cell Transplantation Recipients

Currently, no consensus has been reached on the optimal blood compartment to be used for surveillance of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNAemia. Although several comparative studies have been performed correlating CMV and EBV DNA loads in whole blood (WB) versus plasma, to our knowledge, no studies to date have analyzed the kinetics of both viruses in the 2 blood compartments. In this retrospective noninterventional multicenter cohort study, the kinetics of CMV and EBV DNA in 121 hematopoietic stem cell transplantation (HSCT) recipients were investigated by analyzing in parallel 569 and 351 paired samples from 80 and 58 sequential episodes of CMV and EBV DNAemia, respectively. Unlike previous studies, this study used a single automated molecular method that was CE-marked and Food and Drug Administration-approved for use in quantifying CMV and EBV DNA in both plasma and WB. Furthermore, the complete viral replication kinetics of all episodes (including both the ascending and the descending phases of the active infection) was examined in each patient. The previously observed overall correlation between CMV DNA levels in WB and plasma was confirmed (Spearman's ρ?=?.85; P?<?.001). However, although WB and plasma CMV DNAemia reached peak levels simultaneously, in the ascending phase, the median CMV DNA levels in plasma were approximately 1 log10 lower than WB. Furthermore, in patients who received preemptive therapy, CMV DNA showed a delayed decrease in plasma compared with WB. A lower correlation between EBV DNA levels in plasma versus WB was found (Spearman's ρ?=?.61; P?<?.001). EBV DNA kinetics was not consistent in the 2 blood compartments, mostly due to the lower positivity in plasma. Indeed, in 19% of episodes, EBV DNA was negative at the time of the EBV DNA peak in WB. Our results suggest a preferential use of WB for surveillance of CMV and EBV infection in HSCT recipients.     

Evaluation of a New Commercially Available Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Dengue Virus Infection

We evaluated the FDA-cleared InBios dengue virus (DENV) IgM capture enzyme-linked immunosorbent assay (ELISA) for qualitative detection of anti-DENV IgM antibodies from 79 serum samples obtained from dengue virus-infected patients or suspected dengue cases. The agreement, sensitivity, and specificity of the InBios assay compared to the gold standard in-house DENV IgM capture ELISA were 94, 92, and 94%, respectively. We conclude that the InBios DENV IgM capture ELISA can be effectively used for rapid diagnosis of acute or recent DENV infection.     

Comparison of a Transplant Multiplex Viral Panel on the ICEPlex System with Real-Time PCR for Detection of Cytomegalovirus,Epstein-Barr Virus,and BK Virus in Clinical Specimens

We compared a multiplex viral transplant panel on the ICEPlex system to real-time PCR for the detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV). The sensitivities of the ICEPlex were 83.3%, 95.5%, and 65.5% for the detection of CMV, EBV, and BKV, respectively. Interestingly, the multiplex assay detected dual infections in 16/280 (5.7%) samples tested.     

Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.Members of the Brucella genus are gram-negative, aerobic, nonmotile coccobacilli that can infect a broad range of animal hosts. The genome of Brucella consists of two circular chromosomes, with approximate sizes of 2.1 and 1.2 Mbp (21). Genomic studies have shown such a high degree of genetic similarity among the Brucella spp. (10, 12, 25) that a monospecies designation for the genus has been proposed (33). Because of this conservation of sequence, individual species of Brucella are difficult to differentiate using older molecular techniques, but recent advances, such as multilocus analysis of variable number tandem repeats, have been successfully used to distinguish isolates (2, 9, 17). There are now six recognized Brucella species, which are classically distinguished by their host specificity (9, 21). Three of these species, B. abortus, B. suis, and B. melitensis, are major human pathogens, with B. melitensis being the most prevalent (1, 23).B. abortus, B. melitensis, and B. suis are veterinary pathogens which cause spontaneous abortion in livestock (24) and are also the etiological agents of human brucellosis, which has been described as the most common zoonosis worldwide. Transmission of the disease to humans usually occurs through direct contact with infected animals, consumption of contaminated food, or inhalation of aerosolized particles (23), whereas person-to-person transmission rarely occurs (24).Brucellosis is a severe febrile disease that is rarely fatal, but the ease with which Brucella can be spread as an aerosol makes it an attractive biological weapon. In the 1950s, B. suis became the first biological agent weaponized by the United States (4). Due to their moderate ease of dissemination and low mortality rate, B. abortus, B. melitensis, and B. suis are classified as category B critical biological agents by the Centers for Disease Control and Prevention (CDC) (30).Diagnostic methods for brucellosis rely on serological testing or the isolation and cultivation of the organism from clinical specimens, but these methods can be relatively time-consuming and lack sensitivity and specificity (1). The infectious dose for Brucella in humans is 10 to 100 organisms; consequently, diagnostic laboratory personnel who cultivate these organisms are at significant risk of accidental exposure, and brucellosis is one of the most commonly reported laboratory-acquired infections (11). To minimize the risks associated with handling potentially infectious specimens, molecular diagnostic assays, such as real-time PCR, have been developed for the rapid detection of Brucella spp. in a variety of specimen types (8, 9, 14, 22, 26).The increasing use of molecular diagnostics has resulted in increased numbers of specimens submitted to clinical laboratories and has necessitated automation of the processing procedures (32). Given that DNA extraction methods can influence the sensitivity of real-time PCR assays (6), selection of an optimal extraction method is critical for the laboratory detection of Brucella spp. Relatively few studies have evaluated commercial DNA extraction methods specifically for the recovery of Brucella DNA. One such study, by Queipo-Ortuño et al. (27), compared commercial extraction kits for the recovery of Brucella DNA from spiked serum samples and reported that the UltraClean DNA Blood Spin kit provided optimal results. However, their study evaluated only manual extraction kits, which do not provide the high-throughput extraction capacity that is needed in clinical laboratories. Furthermore, it has been demonstrated that laboratories are likely to receive large numbers of specimens during bioterrorism investigations (13, 15, 18), which suggests the need for an evaluation of automated DNA extraction methods.The purpose of this study was to compare the performances of commercial extraction methods with regard to DNA yield and purity as judged by using Brucella genus- and species-specific real-time PCR assays (14). Six extraction methods were evaluated, representing several of the most popular commercially available methodologies for DNA extraction, including magnetic bead purification, filter membrane purification, and alcohol precipitation. The performance evaluation criteria included residual-viability testing of the DNA extracts, limit of detection studies for three Brucella spp. in phosphate-buffered saline (PBS) suspensions of bacterial cells and dried spiked-swab samples, and comparisons of DNA yields, DNA purity, processing costs and times, and required materials.     

Toward Standardization of BK Virus Monitoring: Evaluation of the BK Virus R-gene Kit for Quantification of BK Viral Load in Urine,Whole-Blood,and Plasma Specimens

Screening of BK virus (BKV) replication is recommended to identify patients at increased risk of BKV-associated diseases. However, the heterogeneity of molecular techniques hinders the establishment of universal guidelines for BKV monitoring. Here we aimed to compare the performance of the CE-marked BK virus R-gene kit (R-gene) to the performance of our in-house assay for quantification of BKV DNA loads (BKVL). A 12-specimen panel from the Quality Control for Molecular Diagnostics (QCMD) organization, 163 urine samples, and 88 paired specimens of plasma and whole blood (WB) from transplant recipients were tested. Both the R-gene and in-house assays showed a good correlation within the QCMD panel (r = 0.995 and r = 0.989, respectively). BKVL were highly correlated between assays, although positive biases were observed with the in-house assay in analysis of urine (0.72 ± 0.83 log₁₀ copies/ml), plasma (1.17 ± 0.63 log₁₀ copies/ml), and WB (1.28 ± 0.37 log₁₀ copies/ml). Recalibration with a common calibrator significantly reduced the bias in comparisons between assays. In contrast, BKVL was underestimated with the in-house PCR in eight samples containing BKV genotype II, presenting point mutations at primer-annealing sites. Using the R-gene assay, plasma and WB specimens were found to be equally suitable for quantification of BKVL, as indicated by the high correlation coefficient (r = 0.965, P < 0.0001). In conclusion, the R-gene assay demonstrated reliable performance and higher accuracy than the in-house assay for quantification of BKVL in urine and blood specimens. Screening of BKV replication by a well-validated commercial kit may enable clinical laboratories to assess viral loads with greater reproducibility and precision.     

Multicenter Comparative Evaluation of Five Commercial Methods for Toxoplasma DNA Extraction from Amniotic Fluid

Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.Molecular methods play an important role in the microbiological diagnosis of infectious diseases due to their high sensitivity and specificity (1, 5). Among these, PCR is now recognized as an essential diagnostic tool for congenital toxoplasmosis (1, 8, 11, 19, 20) as well as for toxoplasmosis in immunocompromised individuals (9, 16) and ocular toxoplasmosis (22, 26). Multicenter comparative evaluations have shown sensitivity differences in the PCR detection of Toxoplasma gondii in amniotic fluid (AF), especially when parasite loads were low (<10 T. gondii cells/ml [T/ml]) (2, 14). Since it has been reported that about half of AF samples infected by T. gondii contain fewer than 10 T/ml (8), it is important to use procedures that allow for the detection of such parasite concentrations.Among the different steps that participate in ensuring a reliable molecular detection method, DNA extraction is crucial (3, 12, 24, 27). Indeed, prior to amplification, biological samples (tissue biopsy samples, body fluid samples, tissue scrapings, etc.) must be prepared not only to extract and concentrate the DNA but also to eliminate proteins, lipids, polysaccharides, and other potential inhibitors of the DNA polymerase. DNA extraction consists of nucleic acid isolation, purification, and concentration in an eluted product, and many commercial systems have now become available, replacing in-house methods in many laboratories (25).One of the general aims of the French National Reference Centre for Toxoplasmosis (Centre National de Référence de la Toxoplasmose) is to determine the best molecular detection strategies for Toxoplasma and to recommend them to diagnosis laboratories. In this sense, a specific objective is to better define the importance of DNA extraction in the whole PCR process. This prompted us to compare the performance of widely used commercial DNA extraction kits for T. gondii detection using a strict experimental protocol. We carried out a prospective and multicenter study to evaluate five commercial DNA extraction procedures, two manual and three automated kits, for the isolation of T. gondii DNA in AF. The yield of DNA extraction was assessed by subsequent DNA amplification, combining two Toxoplasma-specific real-time PCR assays using either TaqMan or fluorescence resonance energy transfer (FRET) probe detection, as well as one human beta-globin PCR and one inhibitor search PCR. To detect fine differences among the extraction methods, we decided to work with low concentrations of the parasite, down to 5 tachyzoites/ml (2, 14).     

Field Evaluation of Dried Blood Spots for Routine HIV-1 Viral Load and Drug Resistance Monitoring in Patients Receiving Antiretroviral Therapy in Africa and Asia

Dried blood spots (DBS) can be used in developing countries to alleviate the logistic constraints of using blood plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) testing, but they should be assessed under field conditions. Between 2009 and 2011, we collected paired plasma-DBS samples from treatment-experienced HIV-1-infected adults in Burkina Faso, Cameroon, Senegal, Togo, Thailand, and Vietnam. The DBS were stored at an ambient temperature for 2 to 4 weeks and subsequently at −20°C before testing. VL testing was performed on the plasma samples and DBS using locally available methods: the Abbott m2000rt HIV-1 test, generic G2 real-time PCR, or the NucliSENS EasyQ version 1.2 test. In the case of virological failure (VF), i.e., a plasma VL of ≥1,000 copies/ml, HIVDR genotyping was performed on paired plasma-DBS samples. Overall, we compared 382 plasma-DBS sample pairs for DBS VL testing accuracy. The sensitivities of the different assays in different laboratories for detecting VF using DBS varied from 75% to 100% for the m2000rt test in labs B, C, and D, 91% to 93% for generic G2 real-time PCR in labs A and F, and 85% for the NucliSENS test in lab E. The specificities varied from 82% to 97% for the m2000rt and NucliSENS tests and reached only 60% for the generic G2 test. The NucliSENS test showed good agreement between plasma and DBS VL but underestimated the DBS VL. The lowest agreement was observed for the generic G2 test. Genotyping was successful for 96/124 (77%) DBS tested, and 75/96 (78%) plasma-DBS pairs had identical HIVDR mutations. Significant discrepancies in resistance interpretations were observed in 9 cases, 6 of which were from the same laboratory. DBS can be successfully used as an alternative to blood plasma samples for routine VL and HIVDR monitoring in African and Asian settings. However, the selection of an adequate VL measurement method and the definition of the VF threshold should be considered, and laboratory performance should be monitored.     

Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid and Inexpensive Detection of Cytomegalovirus DNA in Vitreous Specimens from Suspected Cases of Viral Retinitis

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of cytomegalovirus (CMV) was developed and evaluated. The LAMP assay specifically amplified only CMV DNA, and no cross-reactivity with the DNA of herpes simplex virus type 1, varicella-zoster virus, adenovirus, Aspergillus flavus, or Staphylococcus aureus was observed. The sequences of the LAMP assay-positive CMV products were perfectly (100%) matched with the CMV sequence deposited in the GenBank database. The sensitivity of the LAMP assay was found to be 10 copies/μl of CMV DNA. Vitreous samples from 40 patients with suspected retinitis were subjected to LAMP and real-time PCR for the detection of CMV. Of 40 patients with suspected viral retinitis, 10 tested positive for CMV by the real-time PCR and LAMP assays. A 100% concordance was observed between the results of the two methods. The LAMP assay is a rapid, highly specific, and sensitive method for the diagnosis of retinitis caused by CMV.Viral retinitis is commonly caused by herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), cytomegalovirus (CMV), and occasionally, Epstein-Barr virus (EBV) (7). In patients with atypical features and in the early stages of ocular manifestations, clinical differentiation between cases of retinitis associated with CMV and other herpesvirus infections is often difficult (6). The differentiation of CMV retinitis from HSV and VZV retinitis is very important early in the course of the disease, as the therapeutic agent to be used for treatment differs from virus to virus (7). Conventional methods for the diagnosis of viral retinitis include the detection of viral antigen and virus isolation from intraocular specimens (2). These tests have been shown to have low sensitivities for the detection of viruses and are not currently recommended for use for the diagnosis of viral retinitis (2).PCR has proved to be of great utility for the diagnosis of viral retinitis (3, 4, 5, 6, 8). However, owing to the expensive systems required, PCR is still not a very common diagnostic test. Notomi et al. have reported on a novel nucleic acid amplification assay termed the loop-mediated isothermal amplification (LAMP) assay (10). The assay amplifies the DNA under isothermal conditions (63 to 65°C) with high degrees of specificity, efficiency, and speed. The assay can be conducted in the laboratory in a water bath or heating block (10). Thus, the thermal cycling needs of a PCR are avoided. The assay can be used for the rapid detection of pathogens in peripheral health care settings in developing countries. The present study describes the development and evaluation of a simple and cost-effective LAMP assay for the rapid detection of CMV DNA in patients with viral retinitis.     

An Overview of Epstein-Barr Virus: from Discovery to Future Directions for Treatment and Prevention

Discovered in 1964 by Epstein and Barr, the Epstein-Barr virus (EBV) is widespread in all areas of the world, infecting over 95% of the adult population and earning it the informal name, 'Every Body's Virus'. It was shown to be the causal agent of infectious mononucleosis (IM) in 1968, and many reports followed linking EBV to several other clinical syndromes. In 1970, the virus was shown to be able to immortalize B lymphocytes, which are one of its target cells. This oncogenic potential underlies the role of EBV in Burkitt's lymphoma, post-transplant lymphoproliferative disorders and lymphoma in HIV-infected patients. In addition, EBV can infect epithelial cells in the nasopharynx and is a co-factor for development of nasopharyngeal carcinoma. There is no treatment available for IM to date; however, virus-associated complications may be treated with various modes of immune modulation. Currently, radiotherapy and chemotherapy remain the main treatments for EBV-associated malignancies. Recent evidence has suggested that adoptive therapy through infusions of human leukocyte-associated antigen-matched EBV cytotoxic T cells may form a novel strategy for both prophylaxis and treatment of EBV-induced lymphoproliferative disorders. Additionally, a vaccine based on immunization with a structural antigen, gp350, is under evaluation.     

Development and Evaluation of a Blocking Enzyme-Linked Immunosorbent Assay and Virus Neutralization Assay To Detect Antibodies to Viral Hemorrhagic Septicemia Virus

Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.