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Biomedical subjects

D Thouvenot

Publications and source records attributed to D Thouvenot.

At least 37 records · Page 2Linked to original sources
PubMed2000

Reactivation of acyclovir-resistant thymidine kinase-deficient herpes simplex virus harbouring single base insertion within a 7 Gs homopolymer repeat of the thymidine kinase gene.

HSV infections are treated efficiently and prevented by acyclovir, although resistant strains have been reported. Resistance to acyclovir involves mainly mutations in the viral gene encoding thymidine kinase; mutations may lead to an altered or, more frequently, deficient TK. These acyclovir-resistant TK deficient strains are not able to reactivate from a latent infection in an experimental model, compared to TK positive strains. A case is reported of a bone marrow transplant child who developed HSV infection at 11 days post-transplantation. Acyclovir-resistant HSV 1 was isolated on day 19 post-transplantation. The patient was cured of his infection. A resistant virus was detected 20 months later that harboured the same TK gene mutation as the first resistant virus. This mutation is an insertion of one guanine in a homopolymer repeat of seven guanines located at codon 146 of TK. It has previously been reported and associated with the expression of a deficient TK activity and the ability to reactivate in mice. These results corroborate the clinical relevance of this mutation, which is associated with acyclovir-resistant recurrent infections in humans.

Acyclovir↗
PubMed1999

[Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2)].

Herpes simplex virus (HSV) infections are prevalent worldwide. HSV are characterized by their ability to establish and maintain latent infections that can be reactivated. Various clinical presentations of HSV infections are described. Mostly asymptomatic, these infections may become life-threatening when occurring in neonates or when infecting the central nervous system. Accurate diagnosis of HSV infections is important and PCR is the most sensitive technique for detecting HSV. Type-specific serologies could be particularly useful for seroprevalence rates. Aciclovir is an efficient drug for the treatment of herpes simplex virus infections and resistance to this drug has been reported mainly in immunocompromised patients during the course of aciclovir treatment. There is a variety of potential vaccines for prophylaxis of HSV infection, but no vaccine is now available.

Acyclovir↗
PubMed1999

Optimization of glucosinolate separation by micellar electrokinetic capillary chromatography using a Doehlert's experimental design.

The aim of this study was to optimize by micellar electrokinetic chromatography the separation of four glucosinolates, i.e. sinigrin, glucobrassicin and methoxyglucobrassicin involved in Cruciferae resistance mechanisms and glucotropaeolin used as an internal standard. The separation borate buffer which contained sodium dodecyl sulphate, tetramethylammonium hydroxide and methanol was firstly optimized by using a three variable Doehlert experimental design. The optimum concentrations found enabled, for the first time, to obtain an acceptable resolution between the two indole glucosinolates, glucobrassicin and methoxyglucobrassicin. Modifications of the method such as a capillary pre-rinse with pure borate buffer and a step change in voltage during experiment were performed to improve the resolutions between glucosinolates and to reduce the analysis time. This method was validated by a statistical analysis and showed good linearity, repeatability and reproducibility.

Buffers↗
PubMed1999

Phenotypic and genetic characterization of thymidine kinase from clinical strains of varicella-zoster virus resistant to acyclovir.

Varicella-zoster virus (VZV) is a common herpesvirus responsible for disseminated or chronic infections in immunocompromised patients. Effective drugs such as acyclovir (ACV), famciclovir (prodrug of penciclovir), and foscarnet are available to treat these infections. Here we report the phenotypic and genetic characterization of four ACV-resistant VZV strains isolated from AIDS patients and transplant recipients. Sensitivity to six antiviral drugs was determined by an enzyme-linked immunosorbent assay, viral thymidine kinase (TK) activity was measured by comparing [(3)H]thymidine and 1-beta-D-arabinofuranosyl-[(3)H]thymine as substrates, and the TK gene open reading frame was sequenced. Three strains were found to be TK deficient, and the fourth was a mixed population composed of TK-positive and TK-deficient viruses. Each strain presented a unique TK gene mutation that could account for ACV resistance. In one strain, the deletion of two nucleotides at codon 215 induced a premature stop signal at codon 217. In another strain, a single nucleotide addition at codon 167 resulted in a premature stop signal at codon 206. In both other strains, we identified amino acid substitutions already described in other ACV-resistant VZV strains: either Glu-->Gly at residue 48 or Arg-->Gly at residue 143. According to our work and data previously reported on resistant VZV strains, there are three areas in the TK gene where 71% of the mutations described to date are located. These areas are putative candidates for a genotypic diagnosis of ACV resistance.

Acyclovir↗
PubMed1999

Enterovirus genome detection in wastewater: multi centric evaluation of a commercial kit.

A multi-centric study was carried out in three laboratories, to evaluate the efficiency of a standardized kit for the detection of enterovirus genome in wastewater. Twenty one samples of 20 liters of wastewater were analyzed before and after concentration through glass wool. Each sample was analyzed with the Amplicor kit as well as with techniques developed independently in each laboratory. The results show that the Amplicor kit is well suited to the detection of enterovirus genome in treated wastewater. The results may be compared to those obtained with semi-nested RT-PCR techniques used in each laboratory. However, the Amplicor kit technique is more simple and has the advantage of providing a standardized technique useful for comparative studies. During this work it was observed that the sensitivity of the detection of infectious viruses and virus genome was improved when concentrated samples were used for analysis.

Cell Culture Techniques↗
PubMed1999

Evaluation of combining upper respiratory tract swab samples with cerebrospinal fluid examination for the diagnosis of enteroviral meningitis in children.

In a prospective comparative study, the use of combined analysis of upper respiratory tract swab samples and cerebrospinal fluid (CSF) samples was assessed to improve the detection rate of enteroviral meningitis in children. An enterovirus was detected in 32% of patients with aseptic meningitis when testing CSF samples alone compared with 71.5% when combining CSF and respiratory tract findings. An enterovirus was detected in 17% of respiratory tract samples in an age- and sex-matched control group without meningitis. Thus, combining the examination of upper respiratory tract with CSF findings may improve the detection rate of enteroviral meningitis. Upper respiratory tract samples should be included in the diagnosis scheme to differentiate benign enteroviral meningitis from other life-threatening infections of the central nervous system.

Adolescent↗
PubMed1998

Epidemics of aseptic meningitis due to enteroviruses following national immunization days in Bahrain.

We report two successive epidemics of aseptic meningitis due to enteroviruses (EV) observed after national immunization days against polio. Meningitis due to echovirus 30 occurred from July 1995 to the end of January 1996, mostly among children aged 0-12 years (95.1% of cases), and meningitis due to echovirus 4 occurred from May 1996 to the end of September 1996 in the same age group. There were 286 and 169 cases, respectively. Specimens from several representative cases were sent to the WHO Collaborating Center for Virus Reference and Research Laboratory for serological testing and virus detection, including polymerase chain reaction (PCR) studies. Using those tests, evidence of echovirus 30 infection was found in 44% of the children who were sampled during the first epidemic and 45.5% during the second. During echovirus 30 and echovirus 4 epidemics, a similar decline in the age-specific attack rate from 19.1/10,000 and 10.1/10,000 population aged 12 years to 2.4/10,000 and 3.6/10,000 population aged 13 years was observed, respectively.

Adolescent↗
PubMed1998

[Comparison of the in vitro sensitivity to cidofovir and ganciclovir of clinical cytomegalovirus isolates. Coordinated Action Group 11].

Cidofovir (CDF) or Vistid is a monophosphate nucleoside analogue that inhibits the DNA polymerase of herpes viruses including the cytomegalovirus (CMV). CDF is active on GCV-resistant strains with a mutation on the phosphotransferase gene (UL97). However, DNA polymerase gene mutations that induce resistance to GCV are responsible for cross-resistance to CDF. Resistance phenotypes to GCV and CDF were determined for 57 CMV strains isolated from blood and urine samples. Sixteen strains were recovered after CDF therapy. Of the remaining 41 CDF-naive strains, 34 were susceptible and seven resistant to GCV. Fifty percent inhibitory concentrations (IC50) for CDF were in the 0.2-2.6 microM range for CDF-naive strains susceptible to GCV. For GCV-resistant strains, IC50 values for CDF were < or = 3 microM for strains with a low level of resistance to GCV (GCV IC50 < 30 microM) and > or = 6 microM for three of the five strains with a high level of resistance to GCV (GCV IC50 > or = 30 microM).

Antiviral Agents↗
PubMed1997

Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.

Adenoviridae↗
PubMed1997

Detection of cytomegalovirus in semen from a population of men seeking infertility evaluation.

OBJECTIVE: To determine the incidence of cytomegalovirus in the ejaculates of infertile men who were seropositive for IgG antibodies to cytomegalovirus. DESIGN: Prospective study. PATIENT(S): We tested cytomegalovirus infection in the semen of men participating in an IVF-ET program. MAIN OUTCOME MEASURE(S): IgG and IgM antibodies to cytomegalovirus were measured in sera. We used polymerase chain reaction (PCR) and cell culture to look for both cytomegalovirus DNA and infectious virus in the semen of 70 men with cytomegalovirus-specific antibodies detected in sera. RESULT(S): Of the infertile couples, 13.5% exhibited "mismatching" serology (i.e., detection of IgG antibodies to cytomegalovirus in male serum only and not in female serum) and constituted a potential risk for cytomegalovirus transmission. Cytomegalovrius was identified in the semen of two patients who were positive for IgG antibodies to cytomegalovirus. Cytomegalovirus DNA also was detected in one positive sample after centrifugation through a three-layer Percoll gradient. CONCLUSION(S): Human cytomegalovirus was present in the semen from a population of infertile men. Rapid detection can be achieved by molecular techniques such as PCR combined with a hybridization assay. Even though cytomegalovirus was infrequently detected in semen, these data must be considered in determining the risk of transmission and developmental anomalies in infected fetuses.

Adult↗
PubMed1997

Rapid direct diagnosis of mumps meningitis by ELISA capture technique.

ELISA capture technique (ELISAc) was carried out using a rabbit hyperimmune serum attached to a solid phase for capturing mumps antigens in cerebrospinal fluid (CSF) in patients with meningitis and/or in supernatants of infected Vero cells. A biotin-labelled rabbit serum prepared from the previous serum was added and the reaction was read by an enzymatic (avidine-peroxidase) reaction by automated reading. The cut-off was calculated in 100 CSFs negative for viruses by conventional diagnosis. The specificity was evaluated in Vero cells infected with 22 CSFs collected from vaccinated children (URABE AM9 attenuated vaccine) who developed meningitis. A guinea pig hyperimmune serum confirmed the specificity. Results in culture correlated with the ELISA capture technique (ELISAc). No cross-reactivity was observed with parainfluenza 1, 2, 3 human reference strains. At least 2.5 ngs of purified mumps proteins were detected corresponding to 10(1.5) infectious particles per ml. ELISAc applied directly to 14 CSFs collected from unvaccinated children with meningitis diagnosed five positive cases, whereas in four cases conventional diagnosis had to be undertaken twice. ELISAc permitted the diagnosis of one additional patient. The test can be carried out in 3 h.

Animals↗
PubMed1996

Evaluation and comparison of PCR and hybridization methods for rapid detection of cytomegalovirus in clinical samples.

Rapid diagnosis of cytomegalovirus (CMV) infection may be obtained by molecular techniques, such as the polymerase chain reaction (PCR) and hybridization assays. The optimal technique to detect CMV in clinical samples was assessed. Two different PCR assays were used, targeting either the major immediate early 1 (MIE 1) or the HXLF 4 gene. The PCR products were detected by gel electrophoresis, dot blotting and an easy to use, rapid, solid phase hybridization assay, DNA enzyme immunoassay (DEIA). Standard tissue culture was also used. Cerebrospinal fluids (18), liver biopsies (9) from hepatic transplant recipients, amniotic fluids (7) from mothers with suspected peripartum infection, and samples (6) of miscellaneous origin (brain and fundus biopsy, pericardial and pleural fluid) were tested. Among the 40 samples, CMV was detected in 19 cases. Three were positive by both molecular techniques and tissue culture, 14 by molecular methods and 2 by culture. 16/19 or 9/19 CMV-positive samples were detected by PCR amplification of the HXLF 4 or MIE 1 gene, respectively and 14/16 HXLF 4-positive samples were detected using either dot-blot or DEIA, compared to 9/16 using gel electrophoresis. Thus, the most sensitive assays for the detection of CMV in clinical samples using the methods compared in the current study were PCR amplification of the HXLF 4 gene followed by dot-blot or DEIA hybridization.

Amniotic Fluid↗
PubMed1996

[Resistance of cytomegalovirus to ganciclovir: rapid detection of the mutations 460 of the UL97 phosphotransferase].

The substitution of methionine by either isoleucine or valine at residue 460 in the UL97 phosphotransferase has been shown to be responsible for resistance to ganciclovir (GCV) in 30% of resistant cytomegalovirus (CMV) isolates [4]. These substitutions require one nucleotide change in the gene (G- > T 1 380 and A- > G 1378 respectively). The aim of this study was to develop a discriminative PCR assay for rapid detection of these DNA changes. A PCR assay was duplicated in parallel for each mutation; to detect G- > T 1380 each reaction mixture contained primer VSUL14 and either primer LNW to distinguish wild type residues or LNM to distinguish mutant residues, and for A- > G 1378 primers were VSUL8 and either MCMW to detect wild type sequences or MCMM to detect mutated residues. For optimal discrimination, primers MCMW and MCMM were designed with a mismatch at position 3'-1. The reference strains AD169, Davis and Towne, a laboratory GCV-resistant mutant RCL1.7, and 33 CMV isolates (10 resistant, 2 indetermined and 21 sensitive) were tested by PCR. AD169, Davis and Towne, and 30 isolates were amplified only with non modified primers, and the absence of 460 mutations was confirmed by sequencing. Two isolates P1 and P2, from a transplanted patient were amplified with both MCMM and MCMW: sequencing analysis shown the presence of a mixture of strain, one of them harbouring A- > G 1378 mutation. One resistant strain was amplified neither with MCMM nor with MCMW: a C- > T silent mutation at nt 1368 was present. As sequencing analysis confirmed PCR results, discriminative PCR enables isolates to be rapidly assessed for the presence or absence of 460 mutations. Moreover, it can distinguish Met to Val from Met to Ile mutations, and allows the analysis of mixtures of sensitive ad resistant strains.

AIDS-Related Opportunistic Infections↗
PubMed1996

[Aerosol administration of a replication defective recombinant adenovirus expressing normal human cDNA-CFTR in the respiratory tractus in patients with cystic fibrosis].

At present it is conceivable to think that gene therapy represents a way to treat or even prevent the respiratory manifestations of cystic fibrosis. Consistent to such a concept, there is sufficient evidence that Ad-CFTR, a recombinant replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator cDNA, can vectorize the expression of a functional CFTR (cystic fibrosis transmembrane conductance regulator) to the nasal and airway epithelia. The clinical protocol was designed to assess the safety of single escalating doses of a replication defective adenovirus expressing the cystic fibrosis transmembrane conductance regulator gene (Ad-CFTR) when administered to the tracheobronchial portion of the airways and whether biological efficacy of CFTR delivery could be demonstrated. Six cystic fibrosis patients received nasal instillation and subsequent aerosol (Optineb, Air Liquide, Paris, France) administration of Ad-CFTR the following day. Doses (pfu) applied to the nose were 10(5) (patients SG and PB), 10(7) (patients FP and EP) and 4 x 10(8) (patients DS and FG), while aerosolised doses were 10(7) (patients SG and PB), 10(8) (patients FP and EP) and 5.4 x 10(8) (patients DS and FG), respectively. No acute toxic effects, no increase in the titer of anti-adenovirus antibodies and no spreading or shedding of Ad-CFTR were detected. In one patient Ad-CFTR DNA was found in the urine and blood two days after aerosolisation. Ad-CFTR DNA was detected in nasal and bronchial brush samples, in BAL, in saliva and tonsils 21, 8, 14 and 4 days post virus administration, respectively. Ad-CFTR mRNA (RT-PCR on bronchial cells) and CFTR protein (immunochemistry on nasal and bronchial cells) were detected up to 14 days following Ad-CFTR administration. These results show that the nebulisation of Ad-CFTR is a possible approach for treating the respiratory manifestation of cystic fibrosis.

Adenoviridae↗
PubMed1995

Gavaserin and saltavalin, new peptide antibiotics produced by Bacillus polymyxa.

A strain of Bacillus polymyxa (BPl), isolated from cauliflower seeds, inhibited the growth of microbial phytopathogens. Growth of this strain in liquid medium containing lactose, ammonium sulfate, biotin, and amino acids, resulted in optimal inhibition in vitro. Two new antibacterial substances were isolated and purified from culture broth. Their molecular masses were, respectively, 911 and 903 daltons. The first compound was named gavaserin because it contained glutamic acid, alanine, valine, serine and 2,4-diaminobutyric acid, and octanoic acid. No fatty acid was detected in the second compound, which was named saltavalin because it contained serine, alanine, leucine, threonine, valine, and 2,4-diaminobutyric acid.

Anti-Bacterial Agents↗