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Biomedical subjects

S Billaudel

Publications and source records attributed to S Billaudel.

At least 37 records · Page 2Linked to original sources
PubMed1999

Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA.

To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.

Animals↗
PubMed1998

Efficacy and safety of stavudine and didanosine combination therapy in antiretroviral-experienced patients.

OBJECTIVES: To assess the efficacy, tolerance, and safety of combination antiretroviral therapy with didanosine and stavudine in HIV-infected patients with CD4+ cell counts > 100 x 10(6)/l and HIV plasma RNA > 10(4) copies/ml previously treated with other antiretroviral agents for at least 3 months. DESIGN: In this open, multicentre, non-randomized, Phase II pilot study, adult patients were administered didanosine (200 mg twice daily) plus stavudine (40 mg twice daily) for 6 months. Patients for whom the first regimen had led to undetectable HIV RNA levels were offered a second 6-month course of treatment; those who had achieved insufficient immunological and virological gains in the first 6 months were given a new combination. METHODS: Primary evaluation of efficacy was based on viral load measured by branched DNA second-generation testing (lower limit of detection, 500 copies/ml) and CD4+ cell counts; secondary evaluations included AIDS-defining events and clinical side-effects. RESULTS: Sixty-five patients with median prior antiretroviral therapy of 24 months (65 with zidovudine, 29 with zalcitabine) were included in the study. At baseline, median CD4+ cell count was 198 x 10(6)/l and median plasma HIV RNA was 80000 copies/ml (4.9 log10 copies/ml). In this heavily pretreated population, an increase in the mean CD4+ cell count was observed (+70 x 10(6)/l at 24 weeks). In addition, rapid and prolonged antiviral activity was seen, with a mean maximal decrease of 1.1 log10 copies/ml at week 4, a mean decrease of 0.89 log10 copies/ml at week 24, and a plasma RNA viraemia < 500 copies/ml achieved in 14% of patients at week 24. CONCLUSIONS: Combination therapy with stavudine and didanosine is safe and leads to a sustained antiviral effect, even in patients with prolonged prior antiretroviral exposure and low CD4+ cell counts.

Adult↗
PubMed1998

Reverse transcriptase PCR detection of astrovirus, hepatitis A virus, and poliovirus in experimentally contaminated mussels: comparison of several extraction and concentration methods.

Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (RT-PCR). Shellfish were experimentally contaminated by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues were processed by using borate buffer, glycine solution, saline beef, and saline beef-Freon extraction methods. The viruses were concentrated by precipitation with polyethylene glycol 6000 (PEG 6000) or PEG 8000 or by organic flocculation. RT-PCR was performed with RNA extracts from crude shellfish extracts and concentrates with and without Sephadex LH20 filtration. The glycine solution and borate buffer extraction methods resulted in significantly more RT-PCR-positive samples than the saline beef extraction method. We assessed the efficiency of 20 combinations of extraction and concentration methods. The borate buffer-organic flocculation, borate buffer-PEG 6000, and glycine solution-PEG 6000 combinations gave RT-PCR-positive results for all 27 samples analyzed for the three viruses. Detoxification of the samples by Sephadex LH20 filtration significantly decreased the efficiency of RT-PCR virus detection.

Animals↗
PubMed1998

Simplified reverse transcription polymerase chain reaction procedure with detection by microplate hybridization for routine screening of hepatitis A virus.

Reverse transcription polymerase chain reaction, using either nested or seminested primers, is used extensively for the detection of viruses in small quantities. However, existing methods are prone to false positive reactions. We report here an improved polymerase chain reaction technique based on the use of longer primers (39 nucleotides) with single-step amplification, applied to the detection of hepatitis A in low quantities. While the sensitivity of this technique (10 x the 50% tissue culture infective dose) is equivalent to that of existing methods, it is a simpler procedure, less time consuming, and less susceptible to contamination and therefore provides a more reliable tool for routine diagnosis. Finally, the development of a DNA enzyme immunoassay detection technique and the complete automation of the procedure allow a large number of samples to be processed in clinical laboratories.

Animals↗
PubMed1998

Persistence of infectious hepatitis A virus and its genome in artificial seawater.

The stability of the hepatitis A virus (HAV) genome detectable by RT-PCR in artificial sterile seawater seeded with HAV has been compared to that of HAV detectable in cell culture. The HAV genome was detectable by RT-PCR for 232 days while virus particles were detectable in cell culture for only 35 days. This difference in stability indicates that detection of the HAV genome by RT-PCR is not a reliable indicator of the survival of HAV detectable in cell cultures. However, before these results can be extrapolated to stability in natural seawater, the effect of additional elements in the natural environment, such as bacteria, fungi and suspended matter, on the stability of the HAV genome and cell culture infectious HAV particles, will have to be examined.

Cell Line↗
PubMed1997

Usefulness of DNA viral load quantification for cytomegalovirus disease monitoring in renal and pancreas/renal transplant recipients.

BACKGROUND: The purpose of this prospective study was to evaluate the usefulness of quantifying DNA-cytomegalovirus (CMV) load for the diagnosis and monitoring of CMV disease among renal and pancreas transplant patients under immunosuppressive drugs. METHODS: A longitudinal study was conducted among 34 consecutive, unselected renal and pancreas/renal transplanted patients in our unit. During the first 3 posttransplant months, weekly monitoring of CMV infection and CMV disease was done, involving the determination of viremia by the shell vial assay, qualitative DNAemia by semi-nested polymerase chain reaction (PCR) and quantitative DNAemia by the hybrid capture system (HCS), a new and original hybridization method (337 samples were collected for each test). Qualitative and quantitative DNAemia results were blinded to physicians and three grades of disease were defined according to CMV related symptom occurrence. RESULTS: PCR was the most sensitive (100%) but the least specific (78%) method for the diagnosis of CMV disease. HCS was specific for CMV genome detection, sensitive and reproducible. Blood DNA levels above 60 pg/ml were predictive of severe or moderate CMV disease (sensitivity, 92%; specificity, 100%). A significant decrease in viral load was observed after ganciclovir administration, and a positive PCR or HCS result at the end of the antiviral treatment was associated with relapse of CMV infection or disease. CONCLUSIONS: It is concluded that quantitative DNAemia detection, with this new commercially available method, can predict disease and may be useful for a rational evaluation of ganciclovir preemptive therapy in such patients.

Adolescent↗
PubMed1997

Successful quantification of cytomegalovirus DNA by competitive PCR and detection with capillary electrophoresis.

Human cytomegalovirus (HCMV) is responsible for severe infections in immunocompromised patients. Viral load has recently been identified as one of the major risk factors for subsequent development of HCMV disease. In this context, we developed a protocol allowing rapid, sensitive and precise quantification of HCMV DNA using competitive PCR run to saturation. Long primers were used for amplification, and internal DNA standard was constructed by PCR, with a primer inducing formation of a loop on the target sequence. The obtained fragment differed from the wild one (142 bp) by 6 bp. Quantitative analysis of PCR-amplified HCMV DNA was carried out using an original system combining capillary gel electrophoresis and u.v. detection. This procedure was evaluated on renal transplant recipients, and the results of quantitative PCR were compared with those of viraemia, qualitative DNAemia and HCMV-related symptoms. High levels of HCMV DNA were associated with HCMV-related symptoms, and in all cases a significant decrease of viral load was observed following DHPG treatment. Competitive PCR with capillary electrophoresis detection appears to provide a sensitive quantification method for HCMV DNA in leukocytes and is easily adaptable to routine laboratory use.

Cytomegalovirus↗
PubMed1997

Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry.

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.

Antibodies, Monoclonal↗
PubMed1997

Increased anti-Epstein-Barr virus antibodies in epidermotropic cutaneous T-cell lymphoma: a study of 64 patients.

Epstein-Barr virus (EBV) is often associated with non-Hodgkin's T-cell lymphomas and has recently been found in the lesions of mycosis fungoides and Sézary syndrome. We sought to determine whether the anti-EBV antibody profile was disturbed in mycosis fungoides and Sézary syndrome and whether there are particular profiles characteristic of disease stage. Anti-EBV antibodies (anti-VCA, -EA and -EBNA) were studied in the sera of 64 patients. An immunoenzymatic technique was used, and the results were compared with the same number of age- and sex-matched healthy controls. Patients with mycosis fungoides and Sézary syndrome developed higher anti-VCA antibody titres (median 1200) than controls (median 320). Thirty-seven patients had anti-VCA > or = 1200 vs. 19 controls (P < 0.01). These elevated anti-VCA antibody titres were associated with positive EA in 19 patients versus three controls. No differences were found between the illness stages. Anti-EBV antibodies were most often found in mycosis fungoides and Sézary syndrome when the serological profile was similar to that of cellular immune deficiencies and EBV-related non-Hodgkin's lymphoma. EBV could be involved, either directly on lymphocytes or, more likely, indirectly by chronic antigenic stimulation.

Adult↗
PubMed1995

Colorimetric microtiter plate hybridization assay using monoclonal antibody for detection of an amplified human immunodeficiency virus target.

Since detection of amplified polymerase chain reaction products is a complicated procedure, a colorimetric microtiter plate hybridization assay was developed to automate specific detection of amplified HIV targets. In this assay, hybridization is seen by an antibody reacting selectively with double-stranded DNA. One hundred and ten amplified products detected by a DNA enzyme immunoassay (DEIA) and by classical hybridization with a digoxigenin-labeling probe, detection sensitivities were less than 10 HIV targets per 10(6) cells. This study demonstrates the specificity and sensitivity of DEIA for detecting amplified HIV targets. The use of the same equipment as for ELISA and the complete automation of the procedure allow a large number of samples to be processed in the clinical laboratory.

Adult↗
PubMed1995

Direct sequencing of hepatitis A virus strains isolated during an epidemic in France.

Direct sequencing of PCR products was used to study the VP1 region of the hepatitis A virus (HAV) genome (position 2199 to 2356) of nine strains isolated from human stools collected during a hepatitis A epidemic (western France, 1992), three strains from environmental samples (1990, 1991, and 1992), and two HAV cell culture isolates (the French strain CF53/Lyon and strain CLF). These viruses differed from CF53/Lyon (genotype I) by between 1 and 10.3%, and results indicated the existence of two groups of strains belonging to two different subgenotypes (IA and IB). With this sequencing technique it was possible to monitor the epidemiology of HAV and study its relations.

Adolescent↗
PubMed1995

Clinical and practical value of human cytomegalovirus DNAemia detection by semi-nested PCR for follow-up of BMT recipients.

The aim of this prospective study of 162 recipients of bone marrow transplantation (BMT) was to evaluate the use of DNAemia detection by semi-nested PCR for the diagnosis of human cytomegalovirus (HCMV) infection and HCMV disease. We compared the results obtained for DNAemia with those obtained for viremia, using the shell vial assay. Patients were divided in three groups, according to BMT type (allogeneic or autologous) and date of transplant; 876 DNAemia/viremia pairs were analyzed and the overall concordance between the two tests was 97.5%. Discrepancies between the two tests were essentially due to the earlier positivity of DNAemia. Among the 10 patients with positive DNAemia episodes, 9 developed HCMV disease. DNAemia was more sensitive than viremia for HCMV disease diagnosis, while viremia had a higher positive predictive value. DNAemia appeared easily adaptable to routine laboratory use, less expensive and more informative than viremia. This study shows that DNAemia is a method that can replace viremia detection, allowing HCMV infection and disease follow-up of recipients of allogeneic BMT during the first year after BMT.

Adolescent↗
PubMed1994

Development of RT-semi-nested PCR for detection of hepatitis A virus in stool in epidemic conditions.

The purpose of this study was to determine the efficiency of semi-nested PCR in detecting hepatitis A virus (HAV) RNA. During a 2-year period (1990-1991), HAV RNA was searched for in shellfish from the French Brittany coasts using cRNA and vRNA probes. In January 1992, at the time of a hepatitis A outbreak, 28 stool samples were collected from infected patients (18 adults, 10 children) with anti-HAV IgM. Four samples from subjects with negative HAV serology were used as negative controls. Nucleic acid amplification (reverse-transcription-semi-nested PCR) was performed to detect HAV in stool. HAV RNA was purified by phenol-chloroform extraction and converted to cDNA using reverse transcriptase (Mu-MLV). After amplification, PCR products were visualized on an ethidium-bromide-stained gel and confirmed by hybridization with a specific digoxigenin-labelled oligoprobe. Samples were also studied by molecular hybridization with cRNA and vRNA probes. After onset of the illness, HAV RNA was detected over a longer time period by semi-nested PCR (16/28) than by hybridization (0/28). Even though biological diagnosis of hepatitis A will continue to rely on the detection of anti-HAV IgM, PCR should be useful in certain clinical cases (diagnosis of relapse) and for epidemiological and environmental monitoring of viruses.

Adolescent↗
PubMed1994

[Search of papillomavirus genome in synovial membranes and rheumatoid nodules].

IgG antibodies to rat esophagus stratum corneum are highly specific for rheumatoid arthritis and have been recently shown to be directed against human (pro)filaggrin. Although the mechanisms underlying the loss of tolerance of B lymphocytes for filaggrin remain unelucidated, a role of infection with the human papillomavirus has been suggested on the basis of the following facts: 1) the protein exhibiting the greatest molecular homology with filaggrin is papillomavirus-encoded; 2) human papillomavirus proteins bind to cytoskeletal proteins; 3) human papillomavirus replication is enhanced by friction and rheumatoid lesions predominantly develop in areas subjected to friction, as shown by the distribution of rheumatoid nodules; 4) the rheumatoid pannus resembles a benign tumor. The objectives of this study were to look for the human papillomavirus genome in six synovial membrane specimens and two rheumatoid nodule specimens from rheumatoid arthritis patients with disease durations of more than ten years and in 20 synovial membrane specimens collected during surgical procedures in osteoarthritis patients. The methods used were polymerase chain reaction with papillomavirus-specific primers. The papillomavirus genome was not detected in any of the 28 specimens. A role for the human papillomavirus in the pathogenesis of rheumatoid arthritis cannot be ruled out on the basis of these findings because filaggrin is expressed in other tissues, including the thymic medulla epithelium. In addition, the primers used in this study may have been inappropriate.

Arthritis, Rheumatoid↗
PubMed1993

Detection of human papillomavirus in squamous intraepithelial lesions by consensus and type-specific polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify human papillomavirus (HPV) in 216 cervical biopsy specimens from women referred to the gynecological out-patient unit for colposcopy because of an abnormal smear. HPV DNA was screened using type-specific primers for HPV6, 11, 16, 18, 31 and 33 (TS-PCR) as well as a consensus primer located in the E1 region of the HPV genome (C-PCR). TS-PCR specificity was validated by Southern blot analysis. Low-grade (SIL 1) and high-grade (SIL 2) squamous intraepithelial lesions were found in 165 biopsies. HPV16 detection was better with PCR than Southern blot, particularly for SIL 1 and SIL 2. The fact that 10% of HPV16 (all SIL 2) were not detected by C-PCR indicates that both PCR techniques should be performed. C-PCR also detects uncharacterized HPV types (8.6% prevalence in our results), mainly in SIL 1 and SIL 2. HPV16, the most frequently isolated type (prevalence 21%), was associated with SIL 2 in 83% of cases. A low HPV prevalence was found in specimens without dysplastic cells. These results suggest that PCR may be an important tool for identifying women at risk for developing dysplasia or cervical cancer.

Adult↗
PubMed1993

Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination.

Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts).

Animals↗