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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.

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.

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.

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.

This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae , Mycoplasma pneumoniae , and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 × 10 −3 IFU, 5 × 10 −2 color-changing units, and 1 CFU per reaction tube for C. pneumoniae , M. pneumoniae , and Legionella pneumophila , respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila ; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.

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.

The value of biotinylated Oligonucleotide probes for screening and typing by in situ hybridization of the most frequent genital human papillomavirus infections (HPVs 6,11,16,18, 31, and 33) was assessed. Optimal hybridization conditions were defined on a panel of paraffin-embedded tissue sections previously characterized with HPV full genome probes. Mixtures of oligonucleotides rather than single oligonucleotides were used to improve sensitivity and specificity. All HPV-positive specimens were detected by the screening mixture with a sensitivity and specificity similar to that of full genome probes. Typing mixtures were highly specific for each HPV type. This study confirms the potential of Oligonucleotide probes for detecting and typing HPV infections. © 1995 Wiley-Liss, Inc.

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

This work describes the design and initial evaluation of a commercial test allowing the detection of noroviruses and sapoviruses by reverse transcription-PCR (RT-PCR) in a single tube followed by microplate hybridization, as well as the detection of PCR inhibitors. The test was shown to be broadly reactive (except for Melksham-like strains), sensitive, and specific and thus should be useful for calicivirus detection in clinical practice.

We used a novel type of primer system, a system that uses stair primers, in which the primer sequences are based on consensus sequences in the DNA polymerase gene of herpesvirus to detect herpesviruses by PCR. A single PCR in a single tube detected the six major herpesviruses that infect the central nervous system: herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and human herpesvirus 6 (HHV-6). We used the technique to analyze 142 cerebrospinal fluid (CSF) samples that had been stored at −80°C and compared the results with those obtained previously for the same samples by standard, targeted PCR. Four hundred one targeted PCR tests had been run with the 142 samples to detect HSV-1, HSV-2, CMV, and VZV; screening for EBV and HHV-6 was not prescribed when the samples were initially taken. Eighteen CSF samples tested positive by classic targeted PCR. The herpesvirus consensus PCR detected herpesviruses in 37 samples, including 3 samples with coinfections and 17 viral isolates which were not targeted. Two samples identified as infected by the targeted PCR tested negative by the consensus PCR, and eight samples that tested positive by the consensus PCR were negative by the targeted PCR. One hundred three samples scored negative by both the targeted and the consensus PCRs. This preliminary study demonstrates the value of testing for six different herpesviruses simultaneously by a sensitive and straightforward technique rather than screening only for those viruses that are causing infections as suggested by clinical signs.

To assess the prognostic significance of atypical squamous cells of undetermined significance (ASCUS) using an in situ hybridization (ISH) method for destined cervical cytologic smears and a cocktail of biotinylated DNA probes for human papillomavirus (HPV) 6, 11, 16, 18, 31 and 33.Two HPV DNA probe mixtures were applied to the same smear for the simultaneous detection of high-risk HPV types 16, 18, 31 and 33 and low-risk HPV 6 and 11. ISH was carried out on 192 smears. Among them, 59 showed koilocytosis, 91 ASCUS and 42 normal features.Low-risk HPV types were rarely found and associated mainly with koilocytosis (17%). High rates of potentially oncogenic HPV were detected in ASCUS (41%) and condyloma (73%). In addition, similar levels of positivity were found to be associated with ASCUS when using two probe mixtures specific to high-risk HPV: one included HPV genotypes 16 and 18 and the other, genotypes 31 and 33.HPV DNA typing by ISH on cervical cytologic smears might improve the identification of women at high risk of developing precancerous and cancerous cervical lesions.

The Biohit HPV Screening and Typing kits for in situ hybridization of human papillomavirus (HPV) DNA are now commercially available. The HPV Screening kit contains a cocktail of HPV probes, and the Typing kit contains separated hybridization probes for HPV 6, 11, 16, 18, 31, and 33. They were evaluated by comparison with an in situ hybridization (ISH) method, using the Pathogene HPV probes 6/11, 16/18, 31/33/51. One hundred anogenital biopsies from 78 women and 22 men were tested. Among them, 43% showed normal or inflammatory mucosa, 44%, koilocytosis or mild dysplasia, and 13%, moderate to severe dysplasia. Altogether, 60 specimens were positive with the ISH reference method: 17 with the HPV 6/11 probe, 12 with the HPV 16/18 probe, 16 with the HPV 31/33/51 probe, and 15 had mixed infections. The agreement between the Screening test and the homemade ISH is 91%. The Screening test has a sensitivity of 93% and a specificity of 87%. As for the Biohit Typing test, four false-negative samples, and partial or total discordance in nine and four samples, respectively, were observed when compared to our reference method. Thus the agreement between both typing ISH tests is 92%. The sensitivity of the Biohit Typing test is 93%, and the specificity, 91%. The sensitivity decreases to 72% when the 31 and 33 probes are evaluated separately. The Biohit Screening assay is simple, reliable, reproducible, and suitable for rapid routine screening. The Biohit Typing test allows the detection of a specific type of HPV DNA and also permits, in mixed HPV infection, definition of the type of associated HPV DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

20 guinea pigs were exposed to a broad band noise (120-140 dB SPL) for short durations. The conditions of stimulation were strictly identical for all animals. Cochlear microphonic responses (CM) were recorded with conventional differential electrodes after each exposure, from the first turn of the cochlea. Variations were observed in the degree of CM depression and also in the alteration of the CM transfer function. The origin of this variability is discussed.Variabilite dans la diminution du microphonique cochleaire apres exposition au bruitUn lot de 20 cobayes a ete expose a un bruit a large bande, presente a 120 ou 140 dB SPL, pendant de courtes durees, mais progressives. Les conditions de stimulation etaient strictement controlees et identiques pour tous les animaux. Le potential microphonique (PM) etait enregistre, apres chaque presentation du bruit, dans le premier tour de la cochlee, au moyen d'electrodes differentielles classiques. D'importantes variations etaient observees en ce qui concerne le d...

Meat is a complex medium in which nitrates and nitrites react by processes which are not well known. We do not know how to determine the quantities added, and estimating the residual doses depends on the analytic techniques studied. The analysis begins with extractions and purifications by various techniques described. A brief description is then given of 13 techniques for determining nitrites, 5 of which were applied successfully to meat products, and 18 techniques for determining nitrates, 12 of them suitable for meats. The references are given. The authors conclude by stressing the need for better knowledge of the effect of extraction and preparation techniques in order to give meaning to the results obtained.

The laws by which a number of substances fix nitrogen oxide and sodium nitrite were studied. Nitrogen oxide leads to the formation of compounds of the N O X type, characterised by the following equilibrium constants: 1.93 X 10(-2) for sodium ascorbate, 1.96 X 10(-2) for cysteine, 2.79 X 10(-2) for guanidine, 1.43 X 10(-3) for glutathion, 0.384 for sarcosin, 4.6 X 10(-2) for galacturonic acid, and 1.8 X 10(-3) for neuraminic acid. Mucic, benzoic, lactic and gallic acid do not fix this oxide, nor do glycerol and imidazole. Of all the substances listed above, only ascorbate and cysteine fix nitrite. It was not possible to establish any direct fixing law. This fact, taken with the conditions of the medium, leads to the conclusion that nitrite is fixed only after reduction, in the form of nitrogen oxide NO, according to the laws referred to above.

This report outlines an efficient in situ hybridization method for locating specific mRNAs in tissue cryosections using sulfonated cDNA probes. The method involves chemical modification of DNA probes by insertion of a sulfone radical on cytosine residues, which generates a specific epitope. Sulfonated DNA is then detected by using indirect immunochemical procedure. Alternatively, antibodies conjugated to fluorescein or to alkaline phosphatase were used for mRNA detection. In situ hybridization was developed to study aspects of mesophyll and bundle sheath cell differentiation in maize leaves. Our results indicate that phosphoenolpyruvate carboxylase (PEP C) mRNA is restricted to mesophyll cells, and the nucleus-encoded mRNA of the small subunit (SSU) ribulose 1,5-bisphosphate carboxylase (RuBP C) is limited to the cytosol of bundle sheath cells. Thus, using in situ hybridization, we have demonstrated that the differential distribution of PEP C and RuBP C proteins in the two cell types also reflects the location of their mRNAs. These data imply either a tissue-specific transcriptional regulation or a selective mRNA degradation.

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