Analytical and Clinical Performance of the NeuMoDx™ Platform for Cytomegalovirus and Epstein-Barr Virus Viral Load Testing.

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

A Cell-Based Capture Assay for Rapid Virus Detection.

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

The development of a monolith-based purification process for Orthopoxvirus vaccinia virus Lister strain.

The purification of large viruses remains an important field of research and development. The development of efficient purification trains is restricted by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8µm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1×109pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.

The use of RetroNectin in studies requiring in vitro HIV-1 infection of human hematopoietic stem/progenitor cells.

Human immunodeficiency virus (HIV) causes damage, directly or indirectly, to the whole hematopoietic system, including CD34+ hematopoietic stem/progenitor cells (HSPCs). CXCR4-tropic strains of HIV-1 may affect the function of CD34+CXCR4+ progenitor cells either by infecting the cells or modifying the dynamics of more differentiated hematopoietic cells. However, CD34+ cells are known for their resistance to HIV-1 infection in vitro, which restricts any detailed analysis of the impact of HIV on HSPCs. We report the use of RetroNectin, a recombinant fibronectin fragment used for gene transfer with lentiviral vectors, to overcome the limitation associated with CD34+ cell resistance to HIV-1 infection. RetroNectin coating of plates improved in vitro HIV-1 infectivity on human CD34+ cells by 10 fold. This resulted in stable HIV-1 infection for 5 weeks in an OP9-DL1 coculture. These results suggest that RetroNectin may be a useful tool for long-term monitoring of in vitro HIV-infected CD34+ cells.

Virus purification by CsCl density gradient using general centrifugation.

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Consolidation of molecular testing in clinical virology.

INTRODUCTION: The development of quantitative methods for the detection of viral nucleic acids have significantly improved our ability to manage disease progression and to assess the efficacy of antiviral treatment. Moreover, major advances in molecular technologies during the last decade have allowed the identification of new host genetic markers associated with antiviral drug response but have also strongly revolutionized the way we see and perform virus diagnostics in the coming years. Areas covered: In this review, we describe the history and development of virology diagnostic methods, dedicating particular emphasis on the gradual evolution and recent advances toward the introduction of multiparametric platforms for the syndromic diagnosis. In parallel, we outline the consolidation of viral genome quantification practice in different clinical settings. Expert commentary: More rapid, accurate and affordable molecular technology can be predictable with particular emphasis on emerging techniques (next generation sequencing, digital PCR, point of care testing and syndromic diagnosis) to simplify viral diagnosis in the next future.

Measuring the effectiveness of gaseous virus disinfectants.

The efficacy of gaseous disinfection is critical for prevention and treatment of microbial contamination in biotechnological facilities. For an evaluation of gaseous disinfection efficacy, a down-scaled laboratory model was established, using currently available carrier tests and a custom-made dry fog box. A mixture of peroxyacetic acid and hydrogen peroxide (PAA/HP) was investigated as example, at concentrations between 0.4 and 2.9 mL/m(3) for up to 3 h for inactivation of a panel of lipid-enveloped and non-lipid-enveloped viruses. The influenza viruses were most sensitive to PAA/HP treatment and minute virus of mice was most resistant. Bovine viral diarrhea virus and reovirus III showed intermediate stability and similar inactivation kinetics. Use of the dry fog box circumvents dedicating an entire lab for the investigation, which renders the generation of data more cost-effective and allows for production of highly reproducible kinetic data.

MERS-CoV: consideraciones generales para el diagnóstico por laboratorio / MERS-CoV: general considerations for laboratory diagnosis

La confirmación de un caso probable o sospechoso de infección por MERS-CoV (según criterios clínicos y epidemiológicos), sólo puede ser realizada mediante pruebas de laboratorio. Sin embargo, otros patógenos respiratorios virales (incluyendo, pero no limitado a: Influenza, Virus Sincitial Respiratorio, otros beta y alphacoronavirus humanos comunes) y bacterianos (Streptococcus pneumoniae, Haemophilus influenzae tipo b, Legionella pneumophila) deben ser considerados dentro del algoritmo diagnóstico.

A small volume procedure for viral concentration from water.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

Medical devices; immunology and microbiology devices; classification of John Cunningham Virus serological reagents. Final order.

The Food and Drug Administration (FDA) is classifying John Cunningham Virus (JCV) serological reagents into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

Molecular detection of respiratory viruses.

Over the past several years a wide variety of molecular assays for the detection of respiratory viruses has reached the market. The tests described herein range from kits containing primers and probes detecting specific groups of viruses, to self-contained systems requiring specialized instruments that extract nucleic acids and perform the polymerase chain reaction with little operator input. Some of the tests target just the viruses involved in large yearly epidemics such as influenza, or specific groups of viruses such as the adenoviruses or parainfluenza viruses; others can detect most of the known respiratory viruses and some bacterial agents.

Decrease in hepatitis B prevalence among blood donors in Central-West Brazil

The aim of the present study was to estimate hepatitis B virus seroprevalence among first-time blood donors in the city of Campo Grande, Mato Grosso do Sul State, in the central-western region of Brazil. Findings A retrospective analysis of first-time voluntary blood donor records, from January 2010 to December 2010, was conducted at the Hematology Center of Mato Grosso do Sul. The prevalence of the HBsAg and anti-HBc serological markers and their respective 95% confidence intervals were calculated. Chi-square analysis was performed between the seroprevalence previously found in 2001 and the one determined by the current study. Results were considered statistically significant if p< 0.05. Among 8,840 subjects, 269 (3.04%, 95% CI: 2.7-3.4) were positive for HBV markers. The prevalence rate of HBsAg was 0.19% (95% CI: 0.1-0.3) and anti-HBc alone was 2.85% (95% CI: 2.5-3.2). Conclusions There was no statistically significant difference regarding gender. However, an important association was observed between HBV infection and older age (p< 0.01). The seroprevalence of HBV infection in first-time blood donors diminished from 2001 to 2010 (p< 0.01). Such decrease suggests an improvement in the recruitment of safe donors, the positive impact of vaccination programs and the decreasing of HBV infection prevalence in the general population.

Assembly and characterization of pandemic influenza A H1N1 genome in nasopharyngeal swabs using high-throughput pyrosequencing.

De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.

Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation.

In order to decrease salt crystal formation, reduce the aggregation of viruses in atomic force microscopy (AFM) preparations and obtain an acceptable AFM image, the AFM sampling of viruses must be optimized. In this paper, centrifugal AFM sampling of recombinant adeno-associated virus-2 (rAAV-2) was used. The prepared rAAV-2 virus was imaged by AFM using the tapping mode in air. The results indicate that the rAAV-2 viruses prepared by centrifugal AFM sampling methods could be well imaged by AFM. The rAAV-2 viruses exhibited polymorphous particles in the AFM images. The preliminary off-line section analysis of the individual rAAV-2 virus particle indicated that the half-high widths of the large rAAV-2 particles ranged from 18 to 23 nm, while those of the small rAAV-2 particles ranged from 15 to 17 nm, which is almost in agreement with the results obtained by the off-line particle analysis of the rAAV-2 virus particles. Above all, the aggregation of different-sized rAAV-2 particles was directly imaged by AFM, which verifies the previous speculation that the large number (> ~31 nm) distribution of the mean diameter of rAAV-2 virus particles was probably caused by aggregation of rAAV-2 particles.

Viral RNA extraction for in-the-field analysis.

Retroviruses encode their genetic information with RNA molecules, and have a high genomic recombination rate which allows them to mutate more rapidly, thereby posting a higher risk to humans. One important way to help combat a pandemic of viral infectious diseases is early detection before large-scale outbreaks occur. The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) have been used to identify precisely different strains of some very closely related pathogens. However, isolation and detection of viral RNA in the field are difficult due to the unstable nature of viral RNA molecules. Consequently, performing in-the-field nucleic acid analysis to monitor the spread of viruses is financially and technologically challenging in remote and underdeveloped regions that are high-risk areas for outbreaks. A simplified rapid viral RNA extraction method is reported to meet the requirements for in-the-field viral RNA extraction and detection. The ability of this device to perform viral RNA extraction with subsequent RT-PCR detection of retrovirus is demonstrated. This inexpensive device has the potential to be distributed on a large scale to underdeveloped regions for early detection of retrovirus, with the possibility of reducing viral pandemic events.