Marie-José Sanson-Le Pors

Factors associated with septic shock and mortality in generalized peritonitis: comparison between community-acquired and postoperative peritonitis.

IntroductionThe risk factors associated with poor outcome in generalized peritonitis are still debated. Our aim was to analyze clinical and bacteriological factors associated with the occurrence of shock and mortality in patients with secondary generalized peritonitis.MethodsThis was a prospective observational study involving 180 consecutive patients with secondary generalized peritonitis (community-acquired and postoperative) at a single center. We recorded peri-operative occurrence of septic shock and 30-day survival rate and analyzed their associations with patients characteristics (age, gender, SAPS II, liver cirrhosis, cancer, origin of peritonitis), and microbiological/mycological data (peritoneal fluid, blood cultures).ResultsFrequency of septic shock was 41% and overall mortality rate was 19% in our cohort. Patients with septic shock had a mortality rate of 35%, versus 8% for patients without shock. Septic shock occurrence and mortality rate were not different between community-acquired and postoperative peritonitis. Age over 65, two or more microorganisms, or anaerobes in peritoneal fluid culture were independent risk factors of shock. In the subgroup of peritonitis with septic shock, biliary origin was independently associated with increased mortality. In addition, intraperitoneal yeasts and Enterococci were associated with septic shock in community-acquired peritonitis. Yeasts in the peritoneal fluid of postoperative peritonitis were also an independent risk factor of death in patients with septic shock.ConclusionsUnlike previous studies, we observed no difference in incidence of shock and prognosis between community-acquired and postoperative peritonitis. Our findings support the deleterious role of Enterococcus species and yeasts in peritoneal fluid, reinforcing the need for prospective trials evaluating systematic treatment against these microorganisms in patients with secondary peritonitis.

Failure Of Ganciclovir Treatment Associated With Selection Of A Ganciclovir-resistant Cytomegalovirus Strain In A Lung Transplant Recipient

We report the case of a lung transplant recipient with progressive cytomegalovirus (CMV) disease due to a resistant CMV strain emerging under ganciclovir (GCV) therapy. A discriminative polymerase chain reaction (PCR) assay, designed to detect the resistance-related V460 mutation within the viral enzyme UL97, revealed the presence of a mutated strain in a heterogeneous isolate 51 days after transplantation. The conventional antiviral susceptibility assay had failed to demonstrate resistance to GCV. Under prolonged GCV therapy, the mutated strain dominated the wild-type strain, as shown by the PCR assay. This domination led to laboratory resistance, associated with recurrent fever and progressively severe retinitis. As this discriminative PCR assay was shown to be effective in detecting mutated strains that constitute a minority in the virus load, it should allow better management of patients with CMV disease.

Translation regulation of integrons gene cassette expression by the attC sites

Integron are genetic elements able to carry, capture and shuffle the genes embedded in gene cassettes. The attC recombination sites adopt a stable secondary structure when single‐stranded that is necessary for their recombination. In this study, we evaluated the impact of the structure of the attC site on expression of the 3′ gene in class 1 integrons. This was analysed by substituting the attC of the blaIMP‐8 gene cassette with various mutated attC sites spanning a wide range of sizes and secondary structures, and measuring the integron‐dependent translation of the 3′aac(6′)‐Ib7 gene. In the resulting constructs, the 5′‐attC site differentially affected the expression of the aac(6′)‐Ib7 gene. Contrary to what was expected from their proposed role as Rho‐independent transcription terminators, the transcription of the aac(6′)‐Ib7 gene was not affected by the various attC sites. Mutations of natural sites revealed that destabilization of the potential stem‐loop structure of the attC site in the transcript could enhance the expression of the 3′ gene. In particular, the presence of a translated open reading frame was shown to increase translation of the 3′ gene. These findings might be explained by the capacity of the stem‐loop structures to impede ribosome progression.

Rapid determination of antiviral drug susceptibility of human cytomegalovirus by real-time PCR.

A quantitative real-time PCR-based assay was developed for determination of cytomegalovirus (HCMV) susceptibility to antiviral drugs. After HCMV isolate-growth for 4 days, antiviral drug susceptibility was determined by measuring the reduction of intracellular HCMV DNA in the presence of increasing concentrations of either ganciclovir, or foscarnet or cidofovir. The 50% inhibitory concentration (IC(50)) was the drug concentration that reduced the number of HCMV genome copies by 50%. The IC(50) values were measured for seven HCMV reference strains sensitive or resistant to one or more antiviral drugs. The antiviral susceptibility of 21 HCMV isolates was then tested and the results were consistent with prior determination of their phenotype and/or genotype by plaque reduction assay and sequencing. The real-time PCR susceptibility assay reported here was found to be highly reproducible, simpler to perform than the plaque reduction assay, and amenable to use in the routine diagnostic virology laboratory.

Comparison of sequential cytomegalovirus isolates in a patient with lymphoma and failing antiviral therapy

BACKGROUND Long-term anti-cytomegalovirus (CMV) treatments in immunocompromised patients are hampered by resistance to antiviral drugs. Longitudinal changes in the resistance genotype may depend on changes in selective pressure and the complexity of CMV isolates. OBJECTIVE To evaluate longitudinal changes in the CMV resistance genotype and phenotype along with strain-specific variability in a patient with non-Hodgkins lymphoma in whom successive anti-CMV treatments failed. STUDY DESIGN The resistance phenotype and genotype of seven CMV isolates collected from one patient during a 2-year follow-up period were retrospectively analysed. In parallel, we used glycoprotein B (gB) genotyping, and a- and UL10-13-sequence analysis to study CMV interstrain variability. RESULTS The patient was infected by at least three CMV strains plus variants of the parental strains. Resistance to ganciclovir, cidofovir and foscarnet was successively detected during the follow-up period. UL97 protein kinase changes responsible for resistance to ganciclovir were initially detected at residues 591 and 592, and then at position 594. Decreased sensitivity to foscarnet coincided with the appearance of amino acid substitution N495K in DNA polymerase, whereas cross-resistance to ganciclovir and cidofovir was due to the L501I substitution. CONCLUSIONS The CMV isolates obtained from our patient were complex mixtures of strains. Changes in resistance genotypes depended on resistance selective pressure and were not linked to interstrain variation.

Value of a new rapid non-radioactive sequencing method for analysis of the cytomegalovirus UL97 gene in ganciclovir-resistant strains

Various DNA changes located within a restricted region of the UL97 open reading frame were shown to be associated with the resistance of cytomegalovirus strains to ganciclovir (GCV). In order to analyse this UL97 region in sensitive and GCV-resistant strains, a non-radioactive sequencing assay (Promega, Madison, WI, USA) which combines the dideoxy visualisation by silver-staining of the gel was used. Using this assay, polymerase chain reaction products from results were obtained within 1 day. Point mutations modifying the amino acid sequence of the putative UL97 catalytic site were detected in three isolates. These led to an alanine to valine substitution in residue 594 in one strain with reduced GCV sensitivity, and to a cysteine to glycine substitution in residue 592 in two GCV-resistant isolates. These mutations were different from the DNA changes previously mapped in GCV-resistant laboratory or field strains. No amino acid substitution in the UL97 catalytic site was found in GCV-sensitive isolates. Transfer marker experiments are in progress in order to test the significance of these DNA changes for GCV resistance. This rapid non-radioactive sequencing protocol could be a useful tool for analysing the UL97 region encoding the putative UL97 catalytic site of clinical isolates.

Antiviral activity of ganciclovir and artesunate towards human cytomegalovirus in astrocytoma cells.

Antimalarial drug artesunate inhibits cytomegalovirus (HCMV) replication in human fibroblasts. Astrocytes, the major cell type of the brain, support cytomegalovirus (HCMV) replication. The aim of the study was to assess the antiviral activity of artesunate in astrocytoma cell line U373MG in comparison with ganciclovir. The antiviral concentration inhibiting by 50% (IC(50)) the synthesis of viral DNA was measured by real-time PCR in parallel in U373MG and MRC-5 cells. Reference HCMV strains susceptible and resistant to ganciclovir, and clinical isolates were tested. Ganciclovir and artesunate had similar activity in U373MG cells and MRC-5 fibroblasts. The artesunate IC(50)s in U373MG cells (1.5-2.25 μM) were at least 36-fold lower than the 50% cytotoxicity concentrations. Then, the anti-HCMV activity of artesunate was demonstrated in a cancer cell line.

Recurrent infective endocarditis due to Aggregatibacter actinomycetemcomitans: reinfection or relapse?

Aggregatibacter actinomycetemcomitans is commonly part of the normal microflora of the human upper respiratory tract. It has been implicated in periodontal disease and various infections, particularly endocarditis. We report here what we believe to be the first case of recurrent infective endocarditis due to A. actinomycetemcomitans in a 44-year-old woman occurring 5 years after the initial episode. Genomic analysis proved that the strains were closely related. Despite efficient antibiotic treatment, surgery was necessary for recovery.

Hepatic cytolysis and Hepatitis E Virus infection in HIV-positive patients

Background Hepatitis E is an emerging infection in developed countries and progression to chronic hepatitis has been recently reported in some organ transplant recipients. The prevalence and evolution of hepatitis E in HIVinfected patients are unknown. The aim of the study was to assess hepatitis Ev irus (HEV) infection in HIVinfected patients attending a French Parisian hospital. Methods Out of 1250 HIV-infected patients attending the clinics, 108, with elevated transaminase episodes during the last four years, were included in the study. Two hundred and twelve episodes were recorded and 191 plasma samples (1 to 8 per patient), collected simultaneously to the episodes, were retrospectively tested for the presence of anti-HEV IgM and IgG antibodies and HEV RNA. Results An acute infection, documented by positive tests for anti-HEV IgM, low anti-HEV IgG avidity index (10%) and plasma HEV RNA (genotype 3e), was diagnosed in an homosexual patient with a moderate immunodepression (CD4+ lymphocyte count above 200/mm 3 ). This infection was likely locally acquired. It was benign and resolved within two weeks. No persistent carriage of HEV occurred. In addition, three past infections were evidenced, all of them in patients originating from countries with low socio-economic status. No persistent infection was diagnosed in our cohort. Discussion HEV should be tested in HIV-infected patients with elevated transaminase levels. HEV RNA detection should be used to diagnose the infection and monitor recovery.