Viral metagenomics analysis of kidney donors and recipients: Torque teno virus genotyping and prevalence.

The viral load of the ubiquitous and nonpathogenic torque teno virus (TTV) is associated with the grade of immunosuppression of its host. The development of next-generation sequencing (NGS) allowed to describe the human virome of the blood compartment in transplanted patients, and showed that TTV is the most important part of the virome. This study is a descriptive retrospective pilot study of sequencing plasma samples from 15 matched donors and recipients. After sample processing, nucleic acids were amplified by rolling circle amplification and submitted to NGS by ion proton sequencing technology. Results were analyzed after mapping of reads on the 29 TTV reference genomes and de novo assembling of the reads with MIRA software. The number of TTV species present in donors and recipients was, on average, 12 in donors and 33 in recipients. TTV species predominantly present in donors were TTV-13 and TTV-18; and in recipients were TTV-P15-2, TTV-27, TTV-HD14a, and TTV-22. We highlighted a significant variability in abundance and composition in sequential samples from recipients. Temporal evolution of TTV populations was clearly observed in recipients, but no preferential transmission of a species from donor to recipient was evidenced. Diversity and population expansion were observed in kidney recipients. Further study of TTV species could help assess the potential impact of each species of this virus.

Development of a standardized real time PCR for Torque teno viruses (TTV) viral load detection and quantification: A new tool for immune monitoring.

BACKGROUND: Torque teno viruses (TTV) are small DNA viruses whose replication is closely linked to immune status. A growing number of publications underlined the potential of TTV viral load as an indicator of immunosuppression. OBJECTIVES: To demonstrate the analytical performance of the first standardized RUO (Research Use Only) assay to detect and quantify human TTV DNA in whole blood and plasma. STUDY DESIGN: We established analytical performances for TTV load measurement in various populations. The TTV kinetics were followed in kidney recipients. TTV viral load was analyzed on whole blood samples from 42 kidney recipients follow-up, 53 kidney deceased donors and 31 healthy volunteers. RESULTS: The qPCR TTV assay detects the most prevalent human TTV genotypes and does not cross react with other viruses. Limit of detection was 2.2 log10 copies/mL in whole blood and plasma, linearity and precision were demonstrated over the range 1.61 to 10.61 log10 copies/mL in whole blood. Prevalence of TTV DNA in blood differed significantly among groups: 45% in healthy volunteers, 74% in donors and 83% in kidney recipients. In kidney recipients, early TTV kinetics were comparable to those previously observed with in-house assays in other transplant settings: viral load increased from an average of 4.3 log10 to 7.9 log10 copies/mL within the first 75 days post transplantation. CONCLUSION: This TTV assay showed high analytical sensitivity, specificity, linearity and precision. It is a useful standardized tool to further evaluate TTV load as a biomarker of immune status that could improve individual treatment strategy.

Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples.

BACKGROUND: Sample quality is a fundamental parameter for the successful diagnosis of respiratory viruses. This parameter depends upon the concentration of epithelial cells. Respiratory samples are usually heterogeneous, which makes relative quantification of the viral load, against the quantity of cells, the most suitable measurement. The quantification of viral load in the field of respiratory viruses is a vital piece of information. Quantification is required from RNA or DNA viral genomes extracted. OBJECTIVES: To design (RT-)PCR assays for reference genes, which show stable expression during viral infection, to be used as cellular controls and cellular quantification tools. STUDY DESIGN: Assays were designed for two reference genes: hypoxanthine phosphoribosyltransferase 1 (HPRT1) and ubiquitin C (UBC). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a reference for this study. The transcriptional activity of the three genes was studied during infection with respiratory syncytial virus and adenovirus. The HPRT1 q(RT-)PCR assay was used on clinical samples. RESULTS: All the analysis methods concluded that the three reference genes were stably expressed during viral infection. The HPRT1 q(RT-)PCR assay indicated that the majority of clinical samples (n=301, 69%) had a cellular load of between 100 and 10,000 cells/PCR. The data showed that the concentration decreased as the age of patient increased. CONCLUSIONS: A new tool has been developed and commercialized for quality control and evaluation of cellular concentration in respiratory samples.

A thin layer-based amperometric enzyme immunoassay for the rapid and sensitive diagnosis of respiratory syncytial virus infections.

A simple electrochemical sandwich immunoassay involving a polystyrene microarray slide coated with monoclonal capture antibodies and carbon screen-printed sensors (SPS) was designed for the rapid diagnosis of respiratory syncytial virus (RSV). The detection of the antibody-antigen complex formation relied on the use of a horseradish peroxidase conjugate. Its chronoamperometric measurement detection was performed by confining a droplet of H(2)O(2)/3,3',5,5'-tetramethylbenzidine enzyme substrate/mediator solution within a thin layer between one spot of the microarray and the surface of one screen-printed electrochemical cell. The accumulation of the enzyme product in the thin film of liquid enhanced the electrochemical response which allowed the development of a rapid (25 min) and sensitive thin layer-based amperometric (TLA) enzyme immunoassay. The method was successfully compared to commercially-available immunofluorescent and real-time PCR assays for RSV testing in respiratory secretion clinical samples. This suggests that owing to its rapidity, convenience, low-cost, portability and ability to provide quantified results, the reported concept could be a promising point-of-care diagnostic tool to screen patients with suspected respiratory infection or other types of infectious diseases.

Evaluation of the Epstein-Barr virus R-gene quantification kit in whole blood with different extraction methods and PCR platforms.

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.

New commercially available PCR and microplate hybridization assay for detection and differentiation of human polyomaviruses JC and BK in cerebrospinal fluid, serum, and urine samples.

JC and BK human polyomaviruses (family Polyomaviridae) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.

Development of a PCR-and hybridization-based assay (PCR Adenovirus Consensus) for the detection and the species identification of adenoviruses in respiratory specimens.

BACKGROUND: Antigen detection assays and viral isolation techniques are routinely used to detect adenoviruses (Ad) associated with respiratory infections, and the value of the polymerase chain reaction (PCR) has recently been assessed. OBJECTIVES: This paper describes a PCR-hybridization-immunoenzymatic assay (PCR Adenovirus consensus) used to detect Ad and identify Ad species in respiratory specimens. RESULTS: On seven representative serotypes Ad 12, Ad 3, Ad 7, Ad 11, Ad 1, Ad 8, Ad 4, the mean genome equivalents per ml and the mean 50% infectious doses per ml were 10(6.3)and 10(4), respectively. Using 362 nasal aspirates from children, Ad were detected by immunofluorescence (IF) and culture in 97 cases (27%), by the PCR-Ad hexon method in 107 cases (29.5%) and by the PCR Adenovirus Consensus method in 113 cases (31.2%); 13 samples were found positive by both PCR and negative by the IF and culture methods; five samples were only positive according to the PCR Adenovirus Consensus) method. The sensitivity, specificity, predictive positive value and predictive negative value of the PCR Adenovirus Consensus method were 97.9%, 93.2%, 84%, 99.1%, respectively. The method identified the species (sp) from 91 positive amplicons: 1 Ad sp A, 44 Ad sp B, 42 Ad sp C, 3 Ad sp E, and 1 Ad sp F; 85 isolates were identified by IF or the neutralisation in culture, and 86 by a PCR-RE digestion method. The PCR Adenovirus Consensus detected six positive samples that were negative according to the IF and culture methods, and it identified the precise species of nine IF-positive and culture-negative nasal aspirates. CONCLUSION: The PCR Adenovirus Consensus technique is more efficient than the classical IF or culture techniques for the detection of Ad in respiratory samples. An internal control is included to validate the screening results, and specific probes are used to identify the Ad species.