Détail Unités

AP/HP Pôle 5

- Bactériologie-Virologie-Parasitologie et Hygiène -

Réponses affichées : 436

Impact of 48 week lopinavir/ritonavir monotherapy on blood cell-associated HIV-1-DNA in the MONARK trial

AVETTAND-FENOEL V, FLANDRE P, CHAIX ML, GHOSN J, DELAUGERRE C, RAFFI F, NGOVAN P, COHEN-CODAR I, DELFRAISSY JF, ROUZIOUX C

2010 - J Antimicrob Chemother 65(5):1005-07

Objectives To study the impact of protease inhibitor monotherapy on the HIV-1 blood reservoir in 72 antiretroviral-naive patients. Patients and methods This was evaluated for 72 antiretroviral-naive patients included in the on-treatment analysis of the MONARK trial; 46 patients receiving lopinavir/ritonavir monotherapy and 26 patients receiving a triple therapy. HIV-DNA was quantified in whole blood, using real-time PCR. Results The decrease in HIV-DNA after 48 weeks of lopinavir/ritonavir monotherapy was similar to the decrease observed with triple therapy including lopinavir/ritonavir (-0.77 versus -0.69 log copies/10(6) leucocytes, respectively; P = 0.91). The HIV-DNA decrease was also similar in patients with a virological response in both arms (-0.69 versus -0.69 log copies/10(6) leucocytes, respectively). Interestingly, non-responders had a significantly higher baseline HIV-DNA load than responders in the monotherapy arm; 3.16 versus 2.86 log copies/10(6) leucocytes, respectively (P = 0.05). Conclusions The MONARK data indicate that a lopinavir/ritonavir monotherapy regimen is potent against HIV blood reservoirs in antiretroviral-naive patients after 1 year of treatment, in comparison with a standard-of-care highly active antiretroviral therapy. This impact should be evaluated with other boosted protease inhibitor monotherapies.

Unité(s) : Laboratoire de Microbiologie, EA 3620

[The history of Saint-Martin and Beaumont.]

BERCHE P, LEFRERE JJ

2010 - Presse Med 39(5):598-604

Unité(s) : Laboratoire de Microbiologie

Pneumocystis jirovecii Pneumonia

CATHERINOT E, LANTERNIER F, BOUGNOUX ME, LECUIT M, COUDERC LJ, LORTHOLARY O

2010 - Infect Dis Clin North Am 24(1):107-138

Pneumocystis jirovecii has gained attention during the last decade in the context of the AIDS epidemic and the increasing use of cytotoxic and immunosuppressive therapies. This article summarizes current knowledge on biology, pathophysiology, epidemiology, diagnosis, prevention, and treatment of pulmonary P jirovecii infection, with a particular focus on the evolving pathophysiology and epidemiology. Pneumocystis pneumonia still remains a severe opportunistic infection, associated with a high mortality rate.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses

Granulocyte Colony Stimulating Factor-induced Exacerbation of Fungus-related Immune Restoration Inflammatory Syndrome: A Case of Chronic Disseminated Candidiasis Exacerbation

CHANDESRIS MO, KELAIDI C, MECHAI F, BOUGNOUX ME, BROUSSE N, VIARD JP, POIREE S, LECUIT M, HERMINE O, LORTHOLARY O

2010 - J Microbiol Immunol Infect 43(4):339-343

Chronic disseminated candidiasis is a complication of the intensive therapies of hematological malignancies revealed during hematopoietic recovery, a context reminiscent of the immune restoration inflammatory syndrome in human immunodeficiency virus patients receiving antiretroviral therapy. We report a case of severe exacerbation of chronic disseminated candidiasis after pegylated granulocyte-colony stimulating factor administration. We emphasize the major inflammatory substrate of the disease and suggest that immune-modulating strategies such as hematopoietic growth factors, should be used cautiously in such patients.

Unité(s) : Anatomie Pathologique, Hématologie Adulte, Laboratoire de Microbiologie, Maladies Infectieuses, Radiologie Adulte

La ruse du méningocoque

COUREUIL M, NASSIF X

2010 - M S-Méd. Sci. 26(1):15-17

Parmi les hôtes invisibles du corps humain vivent certains des plus terrifiants pathogènes. S’il faut prendre garde à ses ennemis, il faut aussi se méfier de ses amis. Ainsi, Neisseria meningitidis, une bactérie commensale qui a élu domicile sur la muqueuse nasopharyngée peut s’avérer être un pathogène mortel. N. meningitidis est portée de façon asymptomatique par 3 à 10 % de la population [1] et se transmet d’une personne à une autre par contact direct ou par voie aérienne (gouttelette). Par un mécanisme encore mal connu cette bactérie peut passer dans la circulation sanguine puis franchir la barrière hématoencéphalique (BHE), proliférer dans le liquide céphalorachidien et causer une méningite cérébrospinale. Sa prévalence est de 1 à 25/100 000 habitants selon les pays.

Unité(s) : Laboratoire de Microbiologie, U1002

Evaluation of an upgraded version of the Roche COBAS(R) AmpliPrep/COBAS(R) TaqMan(R) HIV-1 Test for HIV-1 viral load quantification

DAMOND F, AVETTAND-FENOEL V, COLLIN G, ROQUEBERT B, PLANTIER JC, GANON A, SIZMANN D, BABIEL R, GLAUBITZ J, CHAIX ML, BRUN-VEZINET F, DESCAMPS D, ROUZIOUX C

2010 - J Clin Microbiol 48(4):1413-16

We evaluated the performance of the prototype COBAS(R) AmpliPrep/COBAS(R) TaqMan(R) HIV-1 Test, v2.0, using prospective and archived clinical samples initially underquantitated by the COBAS(R) AmpliPrep/COBAS(R) TaqMan(R) HIV-1 Test. The performance of the new test was significantly improved, the majority of underquantitation observed with the first version test was eliminated.

Unité(s) : Laboratoire de Microbiologie, EA 3620

Comparison of antifungal MICs for yeasts obtained using the EUCAST method in a reference laboratory and the Etest in nine different hospital laboratories

DANNAOUI E, PAUGAM A, DEVELOUX M, CHOCHILLON C, MATHERON J, DATRY A, BOUGES-MICHEL C, BONNAL C, DROMER F, BRETAGNE S

2010 - Clin Microbiol Infect 16(7):863-69

Clin Microbiol InfectAbstract In routine laboratory practice, the determination of MICs of antifungals for yeasts often relies on the Etest, because of a good correlation with reference methods. However, this correlation was established through predesigned studies, rather than prospective testing. The surveillance programme of fungaemia (YEASTS programme), implemented since 2003, facilitated our comparison of the Etest and the EUCAST results, obtained on a routine basis in nine different hospitals and in a reference laboratory, respectively. The analysis included 690 isolates recovered from blood culture (362 Candida albicans, 113 Candida glabrata, 69 Candida parapsilosis, 55 Candida tropicalis, 31 Cryptococcus neoformans, and 60 other yeast species) that were tested for their susceptibility to amphotericin B (n = 655), fluconazole (n = 669), itraconazole (n = 198), voriconazole (n = 588), flucytosine (n = 314), and caspofungin (n = 244). Agreement between the Etest and EUCAST datasets was calculated and categorized on the basis of previously published breakpoints. The level of agreement at +/-2 dilutions was 75% for amphotericin B and 90% for flucytosine; for the azoles, it ranged from 71% for itraconazole to 87% for voriconazole. No significant difference was observed among the yeast species, except for Cryptococcus neoformans and flucytosine, with an agreement <40%. Categorical agreement ranged from 60% for itraconazole to 90% for flucytosine. Major and very major discrepancies occurred in <12% and 6%, respectively. The Etest, even when performed on a routine basis, shows a >/=71% agreement with the EUCAST reference method.

Unité(s) : Laboratoire de Microbiologie

Detection of Panton-Valentine toxin in Staphylococcus aureus by mass spectrometry directly from colony: time has not yet come

DAUWALDER O, CARBONNELLE E, BENITO Y, LINA G, NASSIF X, VANDENESCH F, LAURENT F

2010 - Int J Antimicrob Agents 36(2):193-4

Unité(s) : Laboratoire de Microbiologie

Factors predictive of successful darunavir/ritonavir-based therapy in highly antiretroviral-experienced HIV-1-infected patients (the DARWEST study)

DELAUGERRE C, BUYCK JF, PEYTAVIN G, VIARD JP, CHAIX ML, ZUCMAN D, MORTIER E, BLANCHE S, ROUVEIX E, FORCE G, AEGERTER P, DE TRUCHIS P

2010 - J Clin Virol 47(3):248-52

BACKGROUND: Darunavir (DRV) is the latest protease inhibitor (PI) to be approved for antiretroviral-naive and -experienced HIV-infected patients. OBJECTIVES: We examined virologic and immunologic outcomes of highly antiretroviral-experienced patients with triple-class drug resistance receiving DRV/r-based regimens, and attempted to identify factors predictive of virologic success. STUDY DESIGN: We studied patients beginning a ritonavir-boosted DRV (DRV/r 600/100mg twice daily)-containing regimen. Virologic success was defined as plasma viral load (pVL)<50copies/ml at week 36. RESULTS: We studied 62 patients with very severe immunodeficiency (CDC stage C in 69% of cases; median CD4 cell nadir 12/mm(3)). They had previously received a median of four PI and had extensive PI resistance, with a median of three major PI and two DRV resistance mutations. The baseline median pVL and CD4 cell count values were 4.6log(10) and 150/mm(3). At week 36, pVL had fallen by 2.6log(10) and the CD4 cell count had risen by 123cells/mm(3). The virologic success rate was 55% overall, and was improved by concomitant first use of enfuvirtide (67%), raltegravir (69%) or etravirine (75%). Virologic success was independently associated with fewer major PI mutations, previous tipranavir exposure, and concomitant first use of enfuvirtide or raltegravir. CONCLUSIONS: In these highly antiretroviral-experienced patients with triple-class drug resistance, virologic success of DRV-containing regimens was mainly associated with the use of new drug classes and/or fully active drugs. Interestingly, previous tipranavir failure did not undermine the efficacy of DRV, confirming the low level of cross-resistance and, probably, distinct resistance profiles between DRV and tipranavir.

Unité(s) : Immunologie-Hématologie Pédiatriques, Laboratoire de Microbiologie, Maladies Infectieuses

Long-term outcomes in adolescents perinatally infected with HIV-1 and followed up since birth in the French perinatal cohort (EPF/ANRS CO10)

DOLLFUS C, LE CHENADEC J, FAYE A, BLANCHE S, BRIAND N, ROUZIOUX C, WARSZAWSKI J

2010 - Clin Infect Dis 51(2):214-24

BACKGROUND. Increasing numbers of children perinatally infected with human immunodeficiency virus (HIV) are reaching adolescence, largely because of advances in treatment over the past 10 years, but little is known about their current health status. We describe here the living conditions and clinical and immunovirologic outcomes at last evaluation among this pioneering generation of adolescents who were born before the introduction of prophylaxis for vertical transmission and whose infections were diagnosed at a time when treatment options were limited. METHODS. The eligible population consisted of HIV-1-infected children who were born before December 1993 and who were included at birth in the prospective national French Perinatal Cohort (EPF/ANRS CO10). RESULTS. Of the 348 eligible children, 210 (60%; median age, 15 years) were still alive and regularly followed up. Current treatment was highly active antiretroviral therapy (HAART) in 77% and 2 nucleoside analogues in 5.0%; 16% had stopped treatment, and 2% had never been treated. The median CD4 cell count was 557 cells/microL, and 200 cells/microL was exceeded in 94% of patients. The median viral load was 200 copies/mL. Viral load was undetectable in 43% of the adolescents and in 54.5% of those receiving HAART. Median height, weight, and body mass index were similar to French reference values for age, and school achievement was similar to nationwide statistics. Better immunologic status was associated with being younger and with having begun HAART earlier. Undetectable viral load was associated with maternal geographic origin and current HAART. CONCLUSIONS. Given the limited therapeutic options available during the early years of these patients' lives and the challenge presented by treatment adherence during adolescence, the long-term outcomes among this population are encouraging.

Unité(s) : Immunologie-Hématologie Pédiatriques, Laboratoire de Microbiologie, EA 3620

No detection of HIV 1-RNA in semen of men on efficient HAART in the past 4 years of a 2002-2009 survey

DULIOUST E, LERUEZ-VILLE M, GUIBERT J, FUBINI A, JEGOU D, LAUNAY O, SOGNI P, JOUANNET P, ROUZIOUX C

2010 - AIDS 24(10):1595-1598

Unité(s) : Laboratoire de Microbiologie, EA 3620

Unusual presentation of chromoblastomycosis due to Cladophialophora carrionii in a renal and pancreas transplant recipient patient successfully treated with posaconazole and surgical excision

DUPONT C, DUONG TA, MALLET S, MAMZER-BRUNEEL MF, THERVET E, BOUGNOUX ME, DUPONT B

2010 - Transpl Infect Dis 12(2):180-83

C. Dupont, T.A. Duong, S. Mallet, M.F. Mamzer-Bruneel, E. Thervet, M.E. Bougnoux, B. Dupont. Unusual presentation of chromoblastomycosis due to Cladophialophora carrionii in a renal and pancreas transplant recipient patient successfully treated with posaconazole and surgical excision. Transpl Infect Dis 2009. All rights reserved Abstract: Chromoblastomycosis is a chronic, tropical and subtropical, subcutaneous mycosis caused by inoculation of dematiaceous molds. This disease is uncommonly reported in patients who have undergone solid organ transplantation. We describe a case of chromoblastomycosis caused by Cladophialophora carrionii that occurred 7 years after transplantation in a 58-year-old male renal and pancreatic transplant recipient. Diagnosis was based on histopathology and isolation of multiple colonies of the dematiaceous mold in pure culture. Identification was achieved by sequencing of the internal transcribed spacer regions of the rRNA. The patient was successfully treated with posaconazole and surgical excision of a residual lesion.

Unité(s) : Anatomie Pathologique, Dermatologie, Laboratoire de Microbiologie, Maladies Infectieuses, Transplantation Adulte

Identification of clinical coagulase-negative staphylococci, isolated in microbiology laboratories, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and two automated systems

DUPONT C, SIVADON-TARDY V, BILLE E, DAUPHIN B, BERETTI JL, ALVAREZ AS, DEGAND N, FERRONI A, ROTTMAN M, HERRMANN JL, NASSIF X, RONCO E, CARBONNELLE E

2010 - Clin Microbiol Infect 16(7):998-1004

A study was performed to compare matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), linked to a recently engineered microbial identification database, and two rapid identification (ID) automated systems, BD Phoenix (Becton Dickinson Diagnostic Systems, France) and VITEK-2 (bioMerieux, Marcy L'Etoile, France), for the ID of coagulase-negative staphylococci (CoNS). Two hundred and thirty-four clinical isolates of CoNS representing 20 species were analyzed. All CoNS isolates were characterized by sodA gene sequencing, allowing interpretation of the ID results obtained using the respective database of each apparatus. Overall correct ID results were obtained in 93.2%, 75.6% and 75.2% of the cases with the MALDI-TOF-MS, Phoenix and VITEK-2 systems, respectively. Mis-ID and absence of results occurred in 1.7% and 5.1% of the cases with MALDI-TOF-MS, in 23.1% and 1.3% with the Phoenix, and in 13.7% and 0.9% with the VITEK-2 systems, respectively. In addition, with the latter automate, 10.3% of the IDs were proposed with remote possibility. When excluding the CoNS species not included in the databases of at least one of the three systems, the final percentage of correct results, Mis-ID and absence of ID were 97.4%, 1.3% and 1.3% with MALDI-TOF-MS, 79%, 21% and 0% with the Phoenix, and 78.6%, 10.3% and 0.9% with the VITEK-2 system, respectively. The present study demonstrates the robustness and high sensitivity of our microbial identification database used with MALDI-TOF-MS technology. This approach represents a powerful tool for the fast ID of clinical CoNS isolates.

Unité(s) : Laboratoire de Microbiologie

[The role of the microbiologist in the diagnosis of pediatric osteoarticular infections.]

FERRONI A, PEJIN Z, ODENT T, CADILHAC C, BERCHE P, GLORION C

2010 - Arch Pediatr 17(6):766-7

Unité(s) : Laboratoire de Microbiologie, Traumatologie et Orthopédie Pédiatriques

Real time identification of bacteria and yeast in positive blood culture broths by MALDI-TOF-Mass Spectrometry

FERRONI A, SUAREZ S, BERETTI JL, DAUPHIN B, BILLE E, MEYER J, BOUGNOUX ME, ALANIO A, BERCHE P, NASSIF X

2010 - J Clin Microbiol 48(5):1542-48

Delays in identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. MALDI-TOF-MS allows identification of microorganisms directly from colonies within minutes. In this study we have adapted and tested this technology to blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species in negative blood culture bottles. We then tested positive blood cultures or fluids grown in blood culture bottles from patients, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategy. Three hundred and twelve spiked bottles and 434 positive cultures from patients were analysed. Among monomicrobial fluids, MALDI-TOF-MS allowed in twenty minutes a reliable identification at the species, group, genus/family level in 91%, 5% and 2% of cases, respectively. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.

Unité(s) : Laboratoire de Microbiologie

Late postnatal HIV infection in children born to HIV-1-infected mothers in a high-income country

FRANGE P, BURGARD M, LACHASSINNE E, LE CHENADEC J, CHAIX ML, CHAPLAIN C, WARSZAWSKI J, DOLLFUS C, FAYE A, ROUZIOUX C, BLANCHE S

2010 - AIDS 24(11):1771-1776

OBJECTIVE:: To evaluate the risk of late postnatal HIV-1 infection in nonbreastfed children enrolled in the French ANRS Cohort CO01 (EPF). METHODS:: The EPF cohort has prospectively enrolled HIV-infected mother/child pairs with a low proportion of known breastfeeding (<0.2%). Children were followed until they were 24 months old, with HIV-1 DNA/RNA PCR tests performed at birth, M1, M3 and M6 and a late serology at 18-24 months. This substudy included 4539 children who were uninfected at the age of 6 months in 1984-2005. RESULTS:: Five children were late diagnosed with HIV-1 infection despite negative PCR tests until 6 months. In three cases, the infection was diagnosed between 14 and 18 months. The other infections were diagnosed at 10 and 12 years of age because of AIDS-defining symptoms; their last (negative) serologies were performed at 19 and 9 months, respectively. A phylogenetic study performed in the latest case revealed a strong homology between the mother and child strains. No known mode of viral transmission (including breastfeeding or use of premasticated food) could be found. However, we observed previously reported risk factors for intrafamilial HIV-1 transmission: poor socioeconomic backgrounds and sustained HIV-1 viremia in the mothers during the follow-up of their children. CONCLUSION:: Late postnatal HIV-1 infection can rarely be diagnosed in the absence of known breastfeeding in high-income countries. Our results highlight the need for a maintained close follow-up of the noninfected children even after 6 months, especially when there are risk factors for intrafamilial viral transmission.

Unité(s) : Immunologie-Hématologie Pédiatriques, Laboratoire de Microbiologie

Short communication: evidence of HIV type 1 complex and second generation recombinant strains among patients infected in 1997-2007 in France: ANRS CO06 PRIMO Cohort

GALIMAND J, FRANGE P, ROUZIOUX C, DEVEAU C, AVETTAND-FENOEL V, GHOSN J, LASCOUX C, GOUJARD C, MEYER L, CHAIX ML

2010 - AIDS Res Hum Retroviruses 26(6):645-51

Although subtype B strains are still predominant in France, non-B viruses have been isolated from 26% of patients with a primary HIV-1 infection in 2005-2006. The objective of this study was to characterize recombinant-HIV-1 strains by a subtyping based on the phylogenetic analysis of both pol and env sequences. We studied 591 patients who were part of the French PRIMO-Cohort between 1997 and 2007. The RT and V3 regions were sequenced and phylogenetic analyses were performed. Phylogenetic analyses showed concordant subtype results for 91.7% of viruses: 71.6% of the viruses were subtype B and 28.4% belonged to non-B subtypes or circulating recombinant forms (CRFs). Forty-nine strains showed different phylogenies between the two genes (pol and env), indicating that recombinations were observed in 8.3% of the cases. These recombinants were observed in patients from sub-Saharan Africa (28.3%) and in white patients (6.3%). Moreover, among the 49 recombinant viruses, 15 (30.6%) contained a B sequence in the pol or in the env gene compared to 34 (69.4%), which contained non-B or CRF sequences. Twenty-six different recombination patterns involving subtypes, CRFs, or undetermined strains were observed. We have reported the occurrence of new recombinant forms between the two major viral types of strains circulating in France: subtype B and CRF02_AG. Our study confirms a high HIV-1 diversity among patients infected in France within the past 10 years, with a high proportion of non-B strains and the circulation of complex recombinant strains among both sub-Saharan patients and French patients.

Unité(s) : Immunologie-Hématologie Pédiatriques, Laboratoire de Microbiologie, EA 3620

Combined antiretroviral therapy is effective on blood plasma HIV-1-RNA: what about semen HIV-1-RNA levels?

GHOSN J, CHAIX ML

2010 - AIDS 24(2):309-11

Unité(s) : Laboratoire de Microbiologie

Immune and virological benefits of 10 years of permanent viral control with antiretroviral therapy

GUIHOT A, TUBIANA R, BRETON G, MARCELIN AG, SAMRI A, ASSOUMOU L, GONCALVES E, BRICAIRE F, COSTAGLIOLA D, CALVEZ V, ROUZIOUX C, AUTRAN B, KATLAMA C, CARCELAIN G

2010 - AIDS 24(4):614-617

The effects of a 10-year control of HIV replication without viral blips with antiretroviral therapy were examined in progressors. CD4 cell counts did not plateau but showed a continuous increase until the 10th year. Ultrasensitive techniques showed very low plasma HIV RNA and cell-associated DNA levels. Robust memory T cell responses to HIV-p24 were higher than in 3-year treated patients and comparable to those of Elite controllers, whereas interferon-gamma-producing HIV-specific T cells were infrequent. Long-term and efficient antiretroviral therapy provides continuous benefits both on the immune system and on the HIV reservoirs.

Unité(s) : Laboratoire de Microbiologie, EA 3620

Human Listeriosis Caused by Listeria ivanovii

GUILLET C, JOIN-LAMBERT O, MONNIER AL, LECLERCQ A, MECHAI F, MAMZER-BRUNEEL MF, BIELECKA MK, SCORTTI M, DISSON O, BERCHE P, VAZQUEZ-BOLAND J, LORTHOLARY O, LECUIT M

2010 - Emerg Infect Dis 16(1):136-138

Two species of Listeria are pathogenic; L. monocytogenes infects humans and animals, and L. ivanovii has been considered to infect ruminants only. We report L. ivanovii-associated gastroenteritis and bacteremia in a man. This isolate was indistinguishable from prototypic ruminant strains. L. ivanovii is thus an enteric opportunistic human pathogen.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses, Transplantation Adulte

Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection

HOCQUELOUX L, PRAZUCK T, AVETTAND-FENOEL V, LAFEUILLADE A, CARDON B, VIARD JP, ROUZIOUX C

2010 - AIDS 24(10):1598-1601

Five out of 32 patients who received very early and prolonged antiretroviral therapy displayed an unusual, sustained immunovirological control after treatment discontinuation (mean duration: 77 months). These 'post-treatment controllers' did not have the genetic characteristics of spontaneous 'elite' controllers, although they shared very low and stable level of viral reservoir. Treatment may have dramatically decreased this reservoir and preserved potent HIV-specific immunologic responses, inducing a new balance between the virus and the host's immune system in these patients.~~(C) 2010 Lippincott Williams & Wilkins, Inc.~~

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses, EA 3620

Identification of a novel transcriptional regulator involved in pilC1 regulation in Neisseria meningitidis

JAMET A, ROUSSEAU C, MONFORT JB, NASSIF X, MARTIN P

2010 - FEMS Microbiol Lett 304(2):140-147

Abstract Type IV pili are crucial for the virulence of Neisseria meningitidis. PilC proteins belong to the complex protein machinery required for pili biosynthesis. The expression of the pilC1 gene is known to be induced during host cell contact and to be tightly controlled through four promoters, two transcription factors and a two-component signal transduction system. By screening of an insertional-mutant library, we identified a novel regulatory protein, i.e. NMA1805, involved in the pilC1 complex regulation. Increased transcription of gene NMA1805 was shown to result in augmented expression of the pilC1 gene, whereas abrogated expression of gene NMA1805 was associated with an absence of pilC1 induction upon contact with host cells. Moreover, we demonstrated that the NMA1805 gene displayed two promoters. The NMA1805 regulatory protein was evidenced to interact with one of them.

Unité(s) : Laboratoire de Microbiologie, U1002

Mechanisms of meningeal invasion by a bacterial extracellular pathogen, the example of Neisseria meningitidis

JOIN-LAMBERT O, MORAND PC, CARBONNELLE E, COUREUIL M, BILLE E, BOURDOULOUS S, NASSIF X

2010 - Prog Neurobiol 91(2):130-39

The blood-cerebrospinal fluid (CSF) barrier physiologically protects the meningeal spaces from bloodborne bacterial pathogens, due to the existence of specialized junctional interendothelial complexes. A few bacterial pathogens are able to reach the subarachnoidal space and cause bacterial meningitis in humans, a rare but dreadful disease. Surprisingly, most of them are extracellular commensals of the nasopharynx (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae) or of the digestive tract (Escherichia coli and Streptococcus agalactiae). The particular ability of these pathogens to induce meningitis is related to virulence factors that allow them to escape host innate immunity, to multiply within the serum, and to interact closely with the endothelial front line of defense of the blood-CSF barrier. In vitro studies using microvascular brain endothelial cell lines have shown that induced transcytosis may be a common route used by H. influenzae, S. pneumoniae, E. coli and S. agalactiae to reach the CSF. N. meningitidis is a strict human pathogen that interacts very tightly with endothelial cells. Adhesion of the meningococcus is mediated by type IV pili that induce a localized remodeling of the sub cortical cytoskeleton, leading to the formation of endothelial membrane protrusions that anchor bacterial colonies at the endoluminal face of the endothelial cell membrane, allowing a better resistance to blood flow. Recent work has shown that N. meningitidis is also able to recruit the polarity complex Par3/Par6/aPKC that re-routes endothelial cell adhesion molecules of interendothelial junctions, opening a paracellular route for bacteria to cross the endothelial barrier.

Unité(s) : Laboratoire de Microbiologie, U1002

Transient Epstein Barr virus reactivation in CD3 monoclonal antibody-treated patients

KEYMEULEN B, CANDON S, FAFI-KREMER S, ZIEGLER A, LERUEZ-VILLE M, MATHIEU C, VANDEMEULEBROUCKE E, WALTER M, CRENIER L, THERVET E, LEGENDRE C, PIERARD D, HALE G, WALDMANN H, BACH JF, SEIGNEURIN JM, PIPELEERS D, CHATENOUD L

2010 - Blood 115(6):1145-55

Here we report a unique situation in which an early and synchronized EBV reactivation was induced by a 6-day course treatment with a humanized CD3-specific monoclonal antibody in patients with recent onset of type 1 diabetes. The virological and immunological analysis showed that this reactivation was transient, self-limited and isolated, associated with the rapid advent of an EBV-specific T cell response. The anti-CD3 antibody administration was associated with induced short lasting immunosuppression and minor yet clear-cut signs of T cell activation that preceded viral reactivation. Early post-transplant monitoring of renal and islet allograft recipients showed that no comparable phenomenon was observed following administration of full dose immunosuppressive therapy. This EBV reactivation remains of no apparent clinical concern over the long term and should not preclude further development of therapeutic anti-CD3 antibodies. This phenomenon may also direct new research avenues to understand the still ill-defined nature of stimuli triggering EBV reactivation in vivo.

Unité(s) : Laboratoire de Microbiologie, Transplantation Adulte, U1013

Nocardia pseudobrasiliensis as an emerging cause of opportunistic infection after allogeneic haematopoietic stem cell transplantation

LEBEAUX D, LANTERNIER F, DEGAND N, CATHERINOT E, PODGLAJEN I, RUBIO MT, SUAREZ F, LECUIT M, MAINARDI JL, LORTHOLARY O

2010 - J Clin Microbiol 48(2):656-59

We report the case of a 55-year-old man who exhibited a nodular pneumonia four months after an allogeneic haematopoietic stem cell transplantation. Culture of the bronchoalveolar lavage fluid revealed Nocardia pseudobrasiliensis. This recently described carbapenem resistant specie should be included in the differential diagnosis of fungal infection in this setting.

Unité(s) : Laboratoire de Microbiologie, Laboratoire d'Hématologie,Maladies Infectieuses

[Doctor Brown-Sequard's therapy.]

LEFRERE JJ, BERCHE P

2010 - Ann Endocrinol (Paris) 71(2):69-75

Pioneer in the field of hormone therapy, Charles-Edward Brown-Sequard (1817-1894) tried to stop the effects of aging on his contemporaries by injecting animal testicle extracts. His therapy was very popular in the last years of the 19th century. He even had followers in the following century, amongst whom Serge Voronoff (1866-1951), who grafted monkey testicles in replacement of human ones, or Paul Niehans (1882-1971) who practiced therapy using calf embryo cells in Switzerland.

Unité(s) : Laboratoire de Microbiologie

[Andreas Vesalius, pionnier of the human anatomy.]

LEFRERE JJ, BERCHE P

2010 - Presse Med 39(6):713-21

Unité(s) : Laboratoire de Microbiologie

[Laennec invents the stethoscope.]

LEFRERE JJ, BERCHE P

2010 - Presse Med 39(7-8):823-32

Unité(s) : Laboratoire de Microbiologie

Comparison of Early CD4 T-Cell Count in HIV-1 Seroconverters in Cote d'Ivoire and France: The ANRS PRIMO-CI and SEROCO Cohorts

LEWDEN C, THIEBAUT R, BOUFASSA F, COULIBALY A, MALATESTE K, SENG R, TONI TD, INWOLEY A, ROUZIOUX C, MINGA A, ANGLARET X, MEYER L

2010 - J Acquir Immune Defic Syndr 53(2):260-265

OBJECTIVE:: We compared CD4 decline among untreated HIV-1-infected seroconverters living in Cote d'Ivoire (CI) and in France. METHODS:: HIV-1-infected adults were enrolled in the ANRS1220 PRIMO-CI (CI, 1997-2006) and ANRSCO2 SEROCO (France, 1988-1995) cohorts. CD4 count and percentage declines were estimated from enrollment until 24 months of seroconversion by linear random-effect models adjusted for time since seroconversion, age, gender, cell-associated HIV DNA, HIV RNA, and country. RESULTS:: Overall 521 seroconverters (CI 148, 62% men; France 373, 77% men) were enrolled after a median of 7.6 months since seroconversion. Median follow-up duration was 12.7 months. Median age was 28 years. Median baseline CD4 count was 472 and 560 cells per cubic millimeter, respectively. Median baseline HIV RNA was 4.4 and 4.0 log10 copies per milliliter and median HIV DNA was 3.0 and 2.8 log10 copies per 10 peripheral blood mononuclear cells, respectively. In adjusted models, CD4 count and percentage at baseline were lower in CI than in France (P < 0.01), and the difference did not vary during follow-up (P = 0.55). Low HIV RNA and low HIV DNA at baseline were associated with higher CD4 count at baseline. CONCLUSIONS:: CD4 count and percentage were lower in CI than in France. These differences were already observed during early infection and remained similar after adjustment.

Unité(s) : Laboratoire de Microbiologie

Infectious risk associated with arterial catheters compared with central venous catheters

LUCET JC, BOUADMA L, ZAHAR JR, SCHWEBEL C, GEFFROY A, PEASE S, HERAULT MC, HAOUACHE H, ADRIE C, THUONG M, FRANCAIS A, GARROUSTE-ORGEAS M, TIMSIT JF

2010 - Crit Care Med 38(4):1030-1035

BACKGROUND:: Scheduled replacement of central venous catheters and, by extension, arterial catheters, is not recommended because the daily risk of catheter-related infection is considered constant over time after the first catheter days. Arterial catheters are considered at lower risk for catheter-related infection than central venous catheters in the absence of conclusive evidence. OBJECTIVES:: To compare the daily risk and risk factors for colonization and catheter-related infection between arterial catheters and central venous catheters. METHODS:: We used data from a trial of seven intensive care units evaluating different dressing change intervals and a chlorhexidine-impregnated sponge. We determined the daily hazard rate and identified risk factors for colonization using a marginal Cox model for clustered data. RESULTS:: We included 3532 catheters and 27,541 catheter-days. Colonization rates did not differ between arterial catheters and central venous catheters (7.9% [11.4/1000 catheter-days] and 9.6% [11.1/1000 catheter-days], respectively). Arterial catheter and central venous catheter catheter-related infection rates were 0.68% (1.0/1000 catheter-days) and 0.94% (1.09/1000 catheter-days), respectively. The daily hazard rate for colonization increased steadily over time for arterial catheters (p = .008) but remained stable for central venous catheters. Independent risk factors for arterial catheter colonization were respiratory failure and femoral insertion. Independent risk factors for central venous catheter colonization were trauma or absence of septic shock at intensive care unit admission, femoral or jugular insertion, and absence of antibiotic treatment at central venous catheter insertion. CONCLUSIONS:: The risks of colonization and catheter-related infection did not differ between arterial catheters and central venous catheters, indicating that arterial catheter use should receive the same precautions as central venous catheter use. The daily risk was constant over time for central venous catheter after the fifth catheter day but increased significantly over time after the seventh day for arterial catheters. Randomized studies are needed to investigate the impact of scheduled arterial catheter replacement. (Crit Care Med 2010; 38:1030-1035).

Unité(s) : Laboratoire de Microbiologie

Attributable mortality of ventilator-associated pneumonia: respective impact of main characteristics at ICU admission and VAP onset using conditional logistic regression and multi-state models

NGUILE-MAKAO M, ZAHAR JR, FRANCAIS A, TABAH A, GARROUSTE-ORGEAS M, ALLAOUCHICHE B, GOLDGRAN-TOLEDANO D, AZOULAY E, ADRIE C, JAMALI S, CLEC'H C, SOUWEINE B, TIMSIT JF

2010 - Intensive Care Med 36(5):781-9

PURPOSE: Methods for estimating the excess mortality attributable to ventilator-associated pneumonia (VAP) should handle VAP as a time-dependent covariate, since the probability of experiencing VAP increases with the time on mechanical ventilation. VAP-attributable mortality (VAP-AM) varies with definitions, case-mix, causative microorganisms, and treatment adequacy. Our objectives here were to compare VAP-AM estimates obtained using a traditional cohort analysis, a multistate progressive disability model, and a matched-cohort analysis; and to compare VAP-AM estimates according to VAP characteristics. METHODS: We used data from 2,873 mechanically ventilated patients in the Outcomerea database. Among these patients from 12 intensive care units, 434 (15.1%) experienced VAP; of the remaining patients, 1,969 (68.5%) were discharged alive and 470 (16.4%) died. With the multistate model, VAP-AM was 8.1% (95% confidence interval [95%CI], 3.1-13.1%) for 120 days' complete observation, compared to 10.4% (5.6-24.5%) using a matched-cohort approach (2,769 patients) with matching on mechanical ventilation duration followed by conditional logistic regression. VAP-AM was higher in surgical patients and patients with intermediate (but not high) Simplified Acute Physiologic Score II values at ICU admission. VAP-AM was significantly influenced by time to VAP but not by resistance of causative microorganisms. Higher Logistic Organ Dysfunction score at VAP onset dramatically increased VAP-AM (to 31.9% in patients with scores above 7). CONCLUSION: A multistate model that appropriately handled VAP as a time-dependent event produced lower VAP-AM values than conditional logistic regression. VAP-AM varied widely with case-mix. Disease severity at VAP onset markedly influenced VAP-AM; this may contribute to the variability of previous estimates.

Unité(s) : Laboratoire de Microbiologie

[Surgical treatment in the pediatric osteoarticular infections.]

ODENT T, PEJIN Z, CADILHAC C, FERRONI A, GLORION C

2010 - Arch Pediatr 17(6):764-5

Unité(s) : Traumatologie et Orthopédie Pédiatriques, Laboratoire de Microbiologie

Cidofovir may be deleterious in BK virus-associated nephropathy

PALLET N, BURGARD M, QUAMOUSS O, RABANT M, BERERHI L, MARTINEZ F, THERVET E, ANGLICHEAU D, NOEL LH, ROUZIOUX C, LEGENDRE C

2010 - Transplantation 89(12):1542-4

Unité(s) : Anatomie Pathologique, Laboratoire de Microbiologie, Transplantation Adulte

Bilateral urinary leak originating from the native ureters in a dual kidney transplant patient

PALLET N, ROUACH Y, CORREAS JM, ZAHAR JR, LEGENDRE C, MAMZER MF

2010 - Transpl Int 23(9):e51-52

Unité(s) : Laboratoire de Microbiologie, Radiologie Adulte, Transplantation Adulte, Urologie

[HIV infection: Which therapeutical challenges remain in 2009 ?]

ROUZIOUX C

2010 - Ann Pharm Fr 68(1):44-46

More than 25 years after the first description of HIV, a large number of antiretroviral drugs are now available, allowing a major reduction of morbidity and mortality in most developed countries; HIV disease may be now considered as a chronic infection. However, because of the presence of provirus within infected cells totally inaccessible to drugs, treatments need to be maintained for life inducing progressively long terms secondary effects. Researches on reservoirs are a new challenge for the development of new therapeutical approaches. In resource-limited countries, where the epidemics is still going on, with the prevalence of infection that may rise 30 % in some countries such as Swaziland, access to treatments are still very limited. The development of structure including medical laboratories with trained technicians is a large challenge to transfer molecular techniques and diagnostics necessary for therapeutical follow-up of infected adults and children, as well as for the diagnosis of HIV infection in babies born to seropositive mother. French laboratories for medical virology have largely developed many molecular diagnoses for viral infections based on their experience for HIV. H1N1 is now the new challenge.

Unité(s) : Laboratoire de Microbiologie, EA 3620

Bortezomib as the Sole Post-Renal Transplantation Desensitization Agent Does Not Decrease Donor-Specific Anti-HLA Antibodies

SBERRO-SOUSSAN R, ZUBER J, SUBERBIELLE-BOISSEL C, CANDON S, MARTINEZ F, SNANOUDJ R, RABANT M, PALLET N, NOCHY D, ANGLICHEAU D, LERUEZ M, LOUPY A, THERVET E, HERMINE O, LEGENDRE C

2010 - Am J Transplant 10(3):681-86

Persistence of donor-specific anti-HLA antibodies (DSA) associated with antibody-mediated graft injuries following kidney transplantation predicts evolution toward chronic humoral rejection and reduced graft survival. Targeting plasma cells, the main antibody-producing cells, with the proteasome inhibitor bortezomib may be a promising desensitization strategy. We evaluated the in vivo efficacy of one cycle of bortezomib (1.3 mg/m(2)x 4 doses), used as the sole desensitization therapy, in four renal transplant recipients experiencing subacute antibody-mediated rejection with persisting DSA (>2000 [Mean Fluorescence Intensity] MFI). Bortezomib treatment did not significantly decrease DSA MFI within the 150-day posttreatment period in any patient. In addition, antivirus (HBV, VZV and HSV) antibody levels remained stable following treatment suggesting a lack of efficacy on long-lived plasma cells. In conclusion, one cycle of bortezomib alone does not decrease DSA levels in sensitized kidney transplant recipients in the time period studied. These results underscore the need to evaluate this new desensitization agent properly in prospective, randomized and well-controlled studies.

Unité(s) : Hématologie Adulte, Laboratoire d'Immunologie, Laboratoire de Microbiologie, Transplantation Adulte

In untreated HIV-1-infected children, PBMC-associated HIV DNA levels and cell-free HIV RNA levels are correlated to distinct T-lymphocyte populations

SCOTT-ALGARA D, ROUZIOUX C, BLANCHE S, BURGARD M, DIDIER C, RIVIERE Y, BUSEYNE F

2010 - J Acquir Immune Defic Syndr 53(5):553-63

BACKGROUND: Clinical studies support biologically independent roles of cell-free HIV particles and HIV-infected cells in disease progression. The associations between the level of infected cells and immune markers have been poorly studied, particularly in perinatally infected children. OBJECTIVE: We tested the hypothesis that independent roles of cell-free virus and infected cells in HIV pathogenesis should be revealed by different associations between each of them and specific immune markers. METHODS: Levels of HIV RNA and DNA, HIV-specific CD8 T lymphocytes, activated and naive/memory T lymphocytes were determined in 44 untreated HIV-1-infected children. Pearson partial correlation coefficients were used to assess associations between the variables. RESULTS: Here we provide new information, by showing a direct correlation between the percentages of CD4HLA-DR lymphocytes and HIV DNA levels. Furthermore, higher HIV-specific CD8 T-lymphocyte frequencies were associated with lower HIV DNA levels. In contrast, CD838 lymphocytes and memory CD4 lymphocytes were correlated only to the HIV RNA level. All correlations were independent of age and CD4 depletion. CONCLUSIONS: Several immune markers were correlated to either the HIV RNA or the HIV DNA level, but never to both of them, supporting the concept that cell-free virus and infected cells play different roles in HIV-1 immunopathogenesis.

Unité(s) : IMMunologie-Hématologie Pédiatriques, Laboratoire de Microbiologie, Maladies Infectieuses

[Optimized clinical use of vancomycin, a prospective observational study in a Paris teaching hospital.]

TAIEB F, LE MONNIER A, BILLE E, LANTERNIER F, MECHAI F, RIBADEAU-DUMAS F, MAENULEIN E, FORGE C, CORRIOL O, NASSIF X, LORTHOLARY O, ZAHAR JR

2010 - Med Mal Infect 40(5):273-78

INTRODUCTION: Vancomycin is still the cornerstone of antibiotic therapy for patients with suspected or proven invasive methicillin resistant Staphylococcus aureus infections. However, clinical and pharmacodynamic studies underline that appropriate doses depend on the infection site, the patient's weight, his renal function, and the bacterial susceptibility. OBJECTIVE AND METHOD: In this prospective study made in a Paris teaching hospital, our two goals were to describe the modalities of infusion and serum concentration obtained during therapy, in our pediatrics and adults population. RESULTS: In our hospital, vancomycin was administered every eight hours in 83 % (97/102) of the cases and the doses used were 30mg/kg per day in 67 % of cases (68/102). Serum trough levels reached 15mcg/ml and 20mcg/ml in 36 % and 18 % of cases respectively. Moreover, despite adequate doses, trough levels of 15mcg/ml were obtained in only 40 % of cases. CONCLUSION: Vancomycin infusion use could be optimized, by defining optimal serum concentrations and monitoring made by a mobile team of infectious diseases specialists.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses

HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals

TRONO D, VAN LINT C, ROUZIOUX C, VERDIN E, BARRE-SINOUSSI F, CHUN TW, CHOMONT N

2010 - Science 329(5988):174-80

HIV infection can persist in spite of efficacious antiretroviral therapies. Although incomplete inhibition of viral replication may contribute to this phenomenon, this is largely due to the early establishment of a stable reservoir of latently infected cells. Thus, life-long antiviral therapy may be needed to control HIV. Such therapy is prone to drug resistance and cumulative side effects and is an unbearable financial burden for regions of the world hit hardest by the epidemic. This review discusses our current understanding of HIV persistence and the limitations of potential approaches to eradicate the virus and accordingly pleads for a joint multidisciplinary effort toward two highly related goals: the development of an HIV prophylactic vaccine and the achievement of long-term drug-free remissions in HIV-infected individuals.

Unité(s) : Laboratoire de Microbiologie

Duration of colonisation by Enterobacteriaceae producing extended-spectrum beta-lactamase and risk factors for persistent faecal carriage

ZAHAR JR, LANTERNIER F, MECHAI F, FILLEY F, TAIEB F, MAINOT EL, DESCAMPS P, CORRIOL O, FERRONI A, BILLE E, NASSIF X, LORTHOLARY O

2010 - J Hosp Infect 75(1):76-78

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses, Pharmacie

Pharmacokinetics of epinephrine in patients with septic shock: modelization and interaction with endogenous neurohormonal status

ABBOUD I, LEROLLE N, URIEN S, TADIE JM, LEVIEL F, FAGON JY, FAISY C

2009 - Crit Care 13(4):R120

INTRODUCTION: In septic patients, an unpredictable response to epinephrine may be due to pharmacodynamic factors or to non-linear pharmacokinetics. The purpose of this study was to investigate the pharmacokinetics of epinephrine and its determinants in patients with septic shock. METHODS: Thirty-eight consecutive adult patients with septic shock were prospectively recruited immediately before epinephrine infusion. A baseline blood sample (C0) was taken to assess endogenous epinephrine, norepinephrine, renin, aldosterone, and plasma cortisol levels before epinephrine infusion. At a fixed cumulative epinephrine dose adjusted to body weight and under steady-state infusion, a second blood sample (C1) was taken to assess epinephrine and norepinephrine concentrations. Data were analyzed using the nonlinear mixed effect modeling software program NONMEM. RESULTS: Plasma epinephrine concentrations ranged from 4.4 to 540 nmol/L at steady-state infusion (range 0.1 to 7 mg/hr; 0.026 to 1.67 microg/kg/min). A one-compartment model adequately described the data. Only body weight (BW) and New Simplified Acute Physiologic Score (SAPSII) at intensive care unit admission significantly influenced epinephrine clearance: CL (L/hr) = 127 x (BW/70)0.60 x (SAPS II/50)-0.67. The corresponding half-life was 3.5 minutes. Endogenous norepinephrine plasma concentration significantly decreased during epinephrine infusion (median (range) 8.8 (1 - 56.7) at C0 vs. 4.5 (0.3 - 38.9) nmol/L at C1, P < 0.001). CONCLUSIONS: Epinephrine pharmacokinetics is linear in septic shock patients, without any saturation at high doses. Basal neurohormonal status does not influence epinephrine pharmacokinetics. Exogenous epinephrine may alter the endogenous norepinephrine metabolism in septic patients.

Unité(s) : Laboratoire de Microbiologie, EA3620

Model for predicting short-term mortality of severe sepsis

ADRIE C, FRANCAIS A, ALVAREZ-GONZALEZ A, MOUNIER R, AZOULAY E, ZAHAR JR, CLEC'H C, GOLDGRAN-TOLEDANO D, HAMMER L, DESCORPS-DECLERE A, JAMALI S, TIMSIT JF, OUTCOMEREA STUDY G

2009 - Crit Care 13(3):R72

INTRODUCTION: To establish a prognostic model for predicting 14-day mortality in ICU patients with severe sepsis overall and according to place of infection acquisition and to sepsis episode number. METHODS: In this prospective multicentre observational study on a multicentre database (OUTCOMEREA) including data from 12 ICUs, 2268 patients with 2737 episodes of severe sepsis were randomly divided into a training cohort (n = 1458) and a validation cohort (n = 810). Up to four consecutive severe sepsis episodes per patient occurring within the first 28 ICU days were included. We developed a prognostic model for predicting death within 14 days after each episode, based on patient data available at sepsis onset. RESULTS: Independent predictors of death were logistic organ dysfunction (odds ratio (OR), 1.22 per point, P < 10-4), septic shock (OR, 1.40; P = 0.01), rank of severe sepsis episode (1 reference, 2: OR, 1.26; P = 0.10 >or= 3: OR, 2.64; P < 10-3), multiple sources of infection (OR; 1.45, P = 0.03), simplified acute physiology score II (OR, 1.02 per point; P < 10-4), McCabe score ([greater than or equal to]2) (OR, 1.96; P < 10-4), and number of chronic co-morbidities (1: OR, 1.75; P < 10-3, >or= 2: OR, 2.24, P < 10-3). Validity of the model was good in whole cohorts (AUC-ROC, 0.76; 95%CI, 0.74 to 0.79; and HL Chi-square: 15.3 (P = 0.06) for all episodes pooled). CONCLUSIONS: In ICU patients, a prognostic model based on a few easily obtained variables is effective in predicting death within 14 days after the first to fourth episode of severe sepsis complicating community-, hospital-, or ICU-acquired infection.

Unité(s) : Laboratoire de Microbiologie

Genotype of 88 Toxoplasma gondii Isolates Associated with Toxoplasmosis in Immunocompromised Patients and Correlation with Clinical Findings

AJZENBERG D, YERA H, MARTY P, PARIS L, DALLE F, MENOTTI J, AUBERT D, FRANCK J, BESSIERES MH, QUINIO D, PELLOUX H, DELHAES L, DESBOIS N, THULLIEZ P, ROBERT-GANGNEUX F, KAUFFMANN-LACROIX C, PUJOL S, RABODONIRINA M, BOUGNOUX ME, CUISENIER B, DUHAMEL C, DUONG TH, FILISETTI D, FLORI P, GAY-ANDRIEU F, PRATLONG F, NEVEZ G, TOTET A, CARME B, BONNABAU H, DARDE ML, VILLENA I

2009 - J Infect Dis 199(8):1155-1167

We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.

Unité(s) : Laboratoire de Microbiologie

Epstein-Barr Virus Load in Whole Blood Correlates With HIV Surrogate Markers and Lymphoma: A French National Cross-Sectional Study

AMIEL C, LEGOFF J, LESCURE FX, COSTE-BUREL M, DEBACK C, FAFI-KREMER S, GUEUDIN M, LAFON ME, LERUEZ-VILLE M, MENGELLE C, PAYAN C, PILLET S

2009 - JAIDS 50(4):427-429

Unité(s) : Laboratoire de Microbiologie

LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01

AVETTAND-FENOEL V, CHAIX ML, BLANCHE S, BURGARD M, FLOCH C, TOURE K, ALLEMON MC, WARSZAWSKI J, ROUZIOUX C

2009 - J. Med. Virol. 81(2):217-223

HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P < 0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. J. Med. Virol. 81:217-223, 2009. (c) 2008 Wiley-Liss, Inc.

Unité(s) : Laboratoire de Microbiologie, Immuno-Hématologie-Rhumatologie Pédiatriques, EA3620

Universal Antiretroviral Therapy for Pregnant and Breast-Feeding HIV-1-Infected Women: Towards the Elimination of Mother-to-Child Transmission of HIV-1 in Resource-Limited Settings

BECQUET R, EKOUEVI DK, ARRIVE E, STRINGER JS, MEDA N, CHAIX ML, TRELUYER JM, LEROY V, ROUZIOUX C, BLANCHE S, DABIS F

2009 - Clin Infect Dis 49(12):1936-45

Prevention of mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) remains a challenge in most resource-limited settings, particularly in Africa. Single-dose and short-course antiretroviral (ARV) regimens are only partially effective and have failed to achieve wide coverage despite their apparent simplicity. More potent ARV combinations are restricted to pregnant women who need treatment for themselves and are also infrequently used. Furthermore, postnatal transmission via breast-feeding is a serious additional threat. Modifications of infant feeding practices aim to reduce HIV-1 transmission through breast milk; replacement feeding is neither affordable nor safe for the majority of African women, and early breast-feeding cessation (eg, prior to 6 months of life) requires substantial care and nutritional counseling to be practiced safely. The recent roll out of ARV treatment has changed the paradigm of prevention of MTCT. To date, postnatal ARV interventions that have been evaluated target either maternal ARV treatment to selected breast-feeding women, with good efficacy, or single-drug postexposure prophylaxis for short periods of time to their neonates, with a partial efficacy and at the expense of acquisition of drug-related viral resistance. We hypothesize that a viable solution to eliminate pediatric AIDS lies in the universal provision of fully suppressive ARV regimens to all HIV-1-infected women through pregnancy, delivery, and the entire breast-feeding period. On the basis of available evidence, we suggest translating into practice the recently available evidence on this matter without any further delay.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques

L'histoire secrètes des guerres biologiques

BERCHE P

2009 - Ouvrage

Savants fous, médecins tortionnaires ou chefs militaires mégalomanes sont les personnages de ce thriller... où tout est vrai. En matière d'armes biologiques, les États se sont toujours complus dans le mensonge et la désinformation : les armes non conventionnelles, particulièrement dangereuses, ont de quoi terrifier les populations. Le secret qui les entoure permet tout : l'éthique est bafouée au nom de l'efficacité, des expérimentations humaines sont réalisées sous couvert de raison d'État. Des premiers pas de la recherche biologique française pendant la Première Guerre mondiale aux attaques à l'anthrax de 2001, du " cocktail diabolique " américain en pleine Guerre froide aux armes biolétales soviétiques, des expérimentations humaines du Dr Ishii dans les années 1930 aux virus spécifiquement destinés aux populations noires d'Afrique du Sud durant l'apartheid, les récits de L'Histoire secrète des guerres biologiques brossent de notre siècle une fresque d'épouvante. Qui mieux que Patrick Berche, professeur de microbiologie, doyen de la faculté de médecine Paris-Descartes et conseiller du ministre de la Défense pour le risque biologique, pouvait nous dévoiler les arcanes de cette guerre secrète ? Il réussit le tour de force d'être à la fois très documenté dans la recherche et très clair dans l'explication.

Unité(s) : Laboratoire de Microbiologie

Plasma exchange for disseminated cryptococcosis

BOLLEE G, TOUZOT M, MECHAI F, ROYAL V, LEFRERE F, BOUGNOUX ME, DUVIVIER C, VIARD JP, LORTHOLARY O, FAKHOURI F

2009 - Am J Kidney Dis 53(4):673-6

Acute renal failure is frequent in HIV-infected patients and may be related to HIV-associated nephropathy, drugs, or opportunistic infections. We report a peculiar case of disseminated cryptococcosis complicated by acute renal failure associated with obstruction of intrarenal capillaries by Cryptococus neoformans dead bodies and successfully treated with plasma exchanges.

Unité(s) : Maladies Infectieuses, Laboratoire de Microbiologie, Néphrologie Adulte, Anatomie Pathologique, Hématologie Adulte

No relation between in-utero exposure to HAART and intrauterine growth retardation

BRIAND N, MANDELBROT L, LE CHENADEC J, TUBIANA R, TEGLAS JP, FAYE A, DOLLFUS C, ROUZIOUX C, BLANCHE S, WARSZAWSKI J, COHORT AFP

2009 - AIDS 23(10):1235-43

BACKGROUND: The use of HAART during pregnancy is now standard care to prevent mother-to-child HIV transmission in developed countries. There is controversy about its impact on low birth weight. OBJECTIVE: To evaluate the impact of antiretroviral therapy during the pregnancy on birth weight, length and head circumference. METHODS: The study was performed in uninfected infants born to HIV-1-infected mothers, enrolled from 1990 to 2006 in the Agence Nationale de Recherche sur le SIDA French Perinatal Cohort CO1. We excluded mothers who used illicit drugs during pregnancy or had no prenatal care before the third trimester, twins and stillbirths. We used Z-scores adjusted for gestational age and sex. RESULTS: In 8192 mother-infant pairs, the mean birth weight Z-scores increased between 1990 and 1997 and then remained stable until 2006. There was no significant relation between the type of antiretroviral therapy and the proportion of small for gestational age (birth weight Z-score < or = -2SD), which was 4% overall. Infants exposed to HAART compared with monotherapy had a lower mean birth weight Z-scores (difference -0.09, 95% confidence interval -0.15 to -0.02); however, there was no difference between HAART exposure in 2005-2006 and monotherapy in 1999-2004, which corresponded to standard care during each period, respectively. Length or head circumference Z-scores were not associated with antiretroviral therapy exposure. Among pregnancies with HAART, there was no relation between the duration and type of therapy and the anthropometric parameters. CONCLUSION: Our findings in a large cohort suggest that HAART during pregnancy does not increase the incidence of infants who are small for gestational age.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques, EA3620

Factors influencing peripheral blood mononuclear cell-associated HIV-1 DNA level after long-term suppressive antiretroviral therapy in 236 patients

BURGARD M, BOUFASSA F, VIARD JP, GARRIGUE I, RUFFAULT A, IZOPET J, VABRET A, DESCAMPS D, COLSON P, SEIGNEURIN JM, ROUZIOUX C, GROUP AAW

2009 - AIDS 23(16):2165-71

OBJECTIVE: The objectives of this study were to determine whether peripheral blood mononuclear cell (PBMC)-associated HIV-1 DNA level in patients on long-term suppressive antiretroviral therapy (ART) was associated with plasma HIV-1 RNA level, CD4 cell count, and therapeutic factors throughout patient history. DESIGN: Patients receiving triple or dual therapy with plasma HIV-1 RNA below detection limit for more than 3 years were recruited in a multicentric, cross-sectional study within the eight virology laboratories of the Agence Nationale de Recherche sur le SIDA et les Hepatites virales HIV quantification working group, each one in relation with a clinical center. METHODS: PBMC-associated HIV-1 DNA was quantified using a standardized real-time PCR method in all laboratories. RESULTS: A total of 236 patients was included. Median HIV-1 DNA was 2.8 log10 copies/10 PBMCs (interquartile range 2.4-3.0). Univariate analysis showed PBMC HIV-1 DNA level to be related to pre-ART immuno-virologic status (plasma HIV-1 RNA zenith and CD4 cell count nadir) and to current CD4 T-cell count. HIV-1 DNA was lower in patients receiving ART with inferior virologic efficacy, as they also had a higher CD4 nadir and a lower HIV-1 RNA zenith than other patients. PBMC HIV-1 DNA level was not related to therapy duration, to time spent with undetectable HIV-1 RNA or to occurrence of a blip. Plasma HIV-1 RNA zenith and CD4 cell count nadir remained predictive of HIV-1 DNA level in the multivariate model which was associated with 22% of its variability. CONCLUSION: Whatever the duration of treatment, HIV-1 DNA level during ART gives a picture of the intensity of viral replication and immune deficiency reached before starting therapy.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses, EA3620

Early conservative intervention for candida contamination of preservative fluid without allograft nephrectomy

CANAUD G, TIMSIT MO, ZUBER J, BOUGNOUX ME, MEJEAN A, THERVET E, SNANOUDJ R, SBERRO R, MARTINEZ F, LEGENDRE C, MAMZER-BRUNEEL MF

2009 - Nephrol. Dialysis Transplant. 24(4):1325-1327

BACKGROUND: Fungal contamination of kidney allograft preservative fluid can lead to renal arteritis and arterial wall rupture. METHODS: We have evaluated a conservative management strategy based on early antifungal therapy, rigorous morphological monitoring of the graft artery and surgical second look (SSL). Since November 2004, preservative fluid was routinely cultured on specific media for all kidney transplant recipients. RESULTS: In 8/474 cases, results were positive for Candida (albicans 5, glabrata 2, tropicalis 1). Two patients also had candida infection of drainage fluid leading to the diagnosis of operative site infection. Radiological and surgical examinations of the renal graft artery were normal in all cases and nephrectomy was not required. At 12 months, all patients were alive with a functioning allograft. CONCLUSION: Early antifungal therapy with microbiological and morphological follow-up should be recommended as soon as contamination is detected, but SSL is advised only in patients with risk factors for arterial anomalies.

Unité(s) : Laboratoire de Microbiologie, Transplantation Adulte, Urologie

Stable frequency of HIV-1 transmitted drug resistance in patients at the time of primary infection over 1996-2006 in France

CHAIX ML, DESCAMPS D, WIRDEN M, BOCKET L, DELAUGERRE C, TAMALET C, SCHNEIDER V, IZOPET J, MASQUELIER B, ROUZIOUX C, MEYER L, COSTAGLIOLA D, GROUP AAR, COHORT PAC, GROUPS FACS

2009 - AIDS 23(6):717-24

BACKGROUND: Transmission of drug-resistant variants is influenced by several factors, including the HIV-1 RNA levels in HIV-1-infected patients. Our study describes the transmitted drug-resistant virus among 1446 French patients diagnosed at the time of primary infection and included from 1996 to 2006 along with the proportion of chronically infected treated patients in the French Hospital Database on HIV (FHDH). METHODS: Genotypic resistance tests were performed at the time of primary infection. The proportion of patients with viral load <500 copies/ml among treated patients, enrolled in the FHDH, was calculated. RESULTS: Over 1996-2006, the proportion of transmitted resistant viruses to at least one antiretroviral was estimated as 10.9%. When considering class resistance, there was an increase in transmission of nonnucleoside reverse transcriptase inhibitor-resistant virus from 0.6% in 1996-1998 to 4.4% in 1999 (P = 0.034), whereas no change was evidenced for either nucleoside/nucleotide reverse transcriptase inhibitor or protease inhibitor resistance. In the FHDH, the proportion of patients receiving combination antiretroviral therapy (cart) increased from 27.7% in 1996 to 81.4% in 2006 and the proportion of viral load <500 copies/ml in treated patients increased from 17.0% in 1996 to 85.3% in 2006. Phylogenetic analysis revealed that 25.5% of patients harboured HIV-1-non-B virus at the time of primary infection in 2005-2006 compared to 10% in 1996-1998. CONCLUSION: In this large study of patients at the time of primary infection, the frequency of acquired resistant virus was stable over time, over 5% for nucleoside/nucleotide reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor. One explanation for this stability may be the increasing number of treated patients in virological success.

Unité(s) : Laboratoire de Microbiologie, EA3620

Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium

COUREUIL M, MIKATY G, MILLER F, LECUYER H, BERNARD C, BOURDOULOUS S, DUMENIL G, MEGE RM, WEKSLER BB, ROMERO IA, COURAUD PO, NASSIF X

2009 - Science 325(5936):83-7

Type IV pili mediate the initial interaction of many bacterial pathogens with their host cells. In Neisseria meningitidis, the causative agent of cerebrospinal meningitis, type IV pili-mediated adhesion to brain endothelial cells is required for bacteria to cross the blood-brain barrier. Here, type IV pili-mediated adhesion of N. meningitidis to human brain endothelial cells was found to recruit the Par3/Par6/PKCzeta polarity complex that plays a pivotal role in the establishment of eukaryotic cell polarity and the formation of intercellular junctions. This recruitment leads to the formation of ectopic intercellular junctional domains at the site of bacteria-host cell interaction and a subsequent depletion of junctional proteins at the cell-cell interface with opening of the intercellular junctions of the brain-endothelial interface.

Unité(s) : Laboratoire de Microbiologie, U570

Medium-term probability of success of antiretroviral treatment after early warning signs of treatment failure in West African adults

DANEL C, GABILLARD D, INWOLEY A, CHAIX ML, TONI TD, MOH R, MESSOU E, BISSAGNENE E, SALAMON R, EHOLIE S, ANGLARET X

2009 - AIDS Res Hum Retroviruses 25(8):783-93

West African adults with warning signs of failure of antiretroviral treatment (ART) at 6 months were assessed for the probability and factors associated with success at 36 months. After 6 months on ART, patients were included if they had a bad immunologic response (BIR) (month 6 CD4 count < pre-ART CD4 count + 50/mm(3)), incomplete virologic suppression (IVS) (month 6 plasma HIV-1 RNA >300 copies/ml), or both (Dual). They were followed for 30 months after inclusion. CD4 counts and HIV-1 RNA were measured every 3 months. We estimated the probability of reaching immunovirologic success (CD4 count >350/mm(3) and plasma HIV-1 RNA <300 copies/ml) and looked for determinants using Cox analysis. A total of 208 adults were included. Among patients in the IVS and Dual groups, 23% and 38% had at least one genotypic resistance mutation at month 6. The 36-month cumulative probability of immunovirologic success was 0.84 in BIR, 0.81 in IVS, and 0.67 in Dual (p = 0.02). Adjusting for CD4 count, viral load, ART regimen, and morbidity, patients who had no genotypic resistance mutations at month 6 or a medication possession ratio (MPR) >90% between month 6 and month 36 had a likelihood of success 3.8 and 3.6 higher than other patients. The 36-month probability of success was 0.56 and 0.86 in patients with an MPR <90% and >90% and 0.59 and 0.84 in patients with and without resistance. After warning signs of failure at 6 months, a large proportion of patients reaches immunovirologic success before 36 months provided there is a high rate of adherence to medication and the absence of early resistance mutations.

Unité(s) : Laboratoire de Microbiologie

Two-Months-off, Four-Months-on Antiretroviral Regimen Increases the Risk of Resistance, Compared with Continuous Therapy: A Randomized Trial Involving West African Adults

DANEL C, MOH R, CHAIX ML, GABILLARD D, GNOKORO J, DIBY CJ, TONI T, DOHOUN L, ROUZIOUX C, BISSAGNENE E, SALAMON R, ANGLARET X

2009 - J. Infect. Dis. 199(1):66-76

Background. @nbsp; A randomized trial was launched in Cote d'Ivoire in 2002 to compare continuous antiretroviral treatment (hereafter, "C-ART") to an ART regimen of 2 months off and 4 months on therapy (hereafter, "2/4-ART"). We report the final analysis. Methods. @nbsp; A total of 435 adults who were receiving successful ART ((median CD4 cell count prior to ART, 272 cells/mm(3); 88% were receiving a zidovudine-lamivudine-efavirenz regimen) were randomized to receive C-ART or 2/4-ART. The main primary end point was the percentage of patients with <350 CD4 cells/mm(3) at 24 months. The sample size ensured 80% power to demonstrate noninferiority (noninferiority bound, -15%), assuming that 30% of the patients in the C-ART arm would have <350 CD4 cells/mm(3). Other end points were mortality, morbidity, cost of care, genotypic resistance, adherence, and toxicity. Results. @nbsp; The percentage of patients with <350 CD4 cells/mm(3) at 24 months was 5.6% (6 of 107) in the C-ART arm and 14.6% (46 of 315) in the 2/4-ART arm (lower bound of the 95% CI for the difference, -14%). Cost was 18% higher in the C-ART arm, and resistance to nonnucleoside reverse-transcriptase inhibitors (NNRTIs) was 20% higher in the 2/4-ART arm. Other end points were nonconclusive. Conclusions. @nbsp; Although 2/4-ART met the predetermined criteria for noninferiority, the percentage of patients with <350 CD4 cells/mm(3) in the C-ART arm was lower than anticipated, which makes the clinical significance of this noninferiority uncertain. In addition, 2/4-ART led to an unacceptable additional risk of selecting for drug-resistant virus. This new argument against episodic ART strategies is also a caveat against any unplanned ART interruptions in Africa, where most patients receive NNRTIs. Trial registration. @nbsp; ClinicalTrials.gov identifier: NCT00158405 .

Unité(s) : Laboratoire de Microbiologie, EA3620

Perinatal acquisition of drug-resistant HIV-1 infection: mechanisms and long-term outcome

DELAUGERRE C, CHAIX ML, BLANCHE S, WARSZAWSKI J, CORNET D, DOLLFUS C, SCHNEIDER V, BURGARD M, FAYE A, MANDELBROT L, TUBIANA R, ROUZIOUX C, COHORT AFP

2009 - Retrovirology 6(.):85

BACKGROUND: Primary-HIV-1-infection in newborns that occurs under antiretroviral prophylaxis that is a high risk of drug-resistance acquisition. We examine the frequency and the mechanisms of resistance acquisition at the time of infection in newborns. PATIENTS AND METHODS: We studied HIV-1-infected infants born between 01 January 1997 and 31 December 2004 and enrolled in the ANRS-EPF cohort. HIV-1-RNA and HIV-1-DNA samples obtained perinatally from the newborn and mother were subjected to population-based and clonal analyses of drug resistance. If positive, serial samples were obtained from the child for resistance testing. RESULTS: Ninety-two HIV-1-infected infants were born during the study period. Samples were obtained from 32 mother-child pairs and from another 28 newborns. Drug resistance was detected in 12 newborns (20%): drug resistance to nucleoside reverse transcriptase inhibitors was seen in 10 cases, non-nucleoside reverse transcriptase inhibitors in two cases, and protease inhibitors in one case. For 9 children, the detection of the same resistance mutations in mothers' samples (6 among 10 available) and in newborn lymphocytes (6/8) suggests that the newborn was initially infected by a drug-resistant strain. Resistance variants were either transmitted from mother-to-child or selected during subsequent temporal exposure under suboptimal perinatal prophylaxis. Follow-up studies of the infants showed that the resistance pattern remained stable over time, regardless of antiretroviral therapy, suggesting the early cellular archiving of resistant viruses. The absence of resistance in the mother of the other three children (3/10) and neonatal lymphocytes (2/8) suggests that the newborns were infected by a wild-type strain without long-term persistence of resistance when suboptimal prophylaxis was stopped. CONCLUSION: This study confirms the importance of early resistance genotyping of HIV-1-infected newborns. In most cases (75%), drug resistance was archived in the cellular reservoir and persisted during infancy, with or without antiretroviral treatment. This finding stresses the need for effective antiretroviral treatment of pregnant women.

Unité(s) : Immunologie-Hématologie Pédiatriques, Laboratoire de Microbiologie, EA3620

Protease Inhibitors Resistance Analysis in the MONARK Trial Comparing First Line Lopinavir/ritonavir Monotherapy to Lopinavir/ritonavir plus Zidovudine and Lamivudine triple therapy

DELAUGERRE C, FLANDRE P, CHAIX ML, GHOSN J, RAFFI F, DELLAMONICA P, JAEGER H, SHURMANN D, COHEN-CODAR I, NGO VAN P, NORTON M, TABURET AM, DELFRAISSY JF, ROUZIOUX C, FOR THE MSG

2009 - Antimicrob Agents Chemother 53(7):2934-2939

Background: MONARK is a pilot randomized trial comparing the safety and efficacy of lopinavir/ritonavir (LPV/r) monotherapy to LPV/r + zidovudine/lamivudine (triple therapy) in antiretroviral naive HIV-1-infected patients. Methods: Resistance testing was performed at screening and at the time of virological failure (including low-level viremia >50 and <400 copies/mL). Changes from baseline sequences were considered included those of the 2008 IAS-USA resistance protease mutation list. Results: Drug resistance testing was performed for 38 patients (5/53 on triple therapy and 33/83 on monotherapy). In the monotherapy arm until week (W) 96, 18/33 patients showed changes from baseline of whom 5 had virus with major protease inhibitor (PI)-associated resistance mutations (M46I at W40, L76V at W48, M46I+L76V at W48, L10F+V82A at W72, and L76V at W84). Screening and failure phenotypes were available for 4 patients with major PI mutations with a mean LPV IC50 increase of 2.2 fold (0.75 to 4.6). All 3 patients who developed L76V PI mutation were infected with HIV-1 CFR02_AG. In the triple therapy until W48, no major PI mutation was selected among the 3 patients with protease changes. No association between baseline CD4 and viral load, nor week 4 and latest viral load, nor latest LPV trough concentration and emergence of major PI mutation was found. Conclusions: Major PI-associated resistance mutations were evidenced in 5 out of 83 (6%) patients treated with LPV/r monotherapy, suggesting that LPV/r monotherapy is considered an inappropriate first option. The mutation L76V might be considered in lopinavir resistance in further studies.

Unité(s) : Laboratoire de Microbiologie, EA3620

Loss of heterozygosity in commensal isolates of the asexual diploid yeast Candida albicans

DIOGO D, BOUCHIER C, D'ENFERT C, BOUGNOUX ME

2009 - Fungal Genet Biol 46(2):159-68

Candida albicans is a commensal and the most frequent fungal pathogen of humans. One mechanism of genetic variation in this diploid asexual yeast involves loss of heterozygosity (LOH). LOH events occur upon infection and contribute to the acquisition of antifungal resistance in patients. In contrast, little is known about the nature and extent of LOH events during commensalism. Using a combination of single nucleotide polymorphism typing, positional transcript profiling and karyotyping, we have characterized related C. albicans commensal isolates that differ by LOH events. Most of these LOH events encompassed the entirety of the chromosome or a large region extending to the telomere, suggesting chromosome loss or mitotic recombination/break-induced replication events, respectively. They were frequently accompanied by karyotype alterations such as chromosome length polymorphism and copy number variations at other chromosomes. These results demonstrate the high plasticity of the C. albicans genome during commensalism.

Unité(s) : Laboratoire de Microbiologie

Synergistic effect of carbapenem-teicoplanin combination during severe Rhodococcus equi pneumonia in a kidney transplant recipient

EL KAROUI K, GUILLET C, SEKKAL N, LANTERNIER F, MECHAI F, HUE K, HIESSE C, MAMZER-BRUNEEL MF, CATHERINOT E, VIARD JP, MAINARDI JL, LECUIT M, FERRONI A, LORTHOLARY O

2009 - Transpl Infect Dis 11(4):359-62

Rhodococcus equi is a bacterial pathogen of domestic animals that can infect immunocompromised patients, especially those with impaired cellular immunity, such as transplant recipients. No standard treatment has been established, but therapy must be prolonged, as relapses are common and can occur at the initial site or distant locations. Here we report a case of R. equi-associated pulmonary abscess in a renal transplant recipient successfully treated with a combination of carbapenem and teicoplanin. This combination was shown to be synergistic. It has minimal side effects in transplant recipients and appears to be an effective initial treatment for this severe infection.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses, Transplantation Adulte

Effect of mutator P. aeruginosa on antibiotic resistance acquisition and respiratory function in cystic fibrosis

FERRONI A, GUILLEMOT D, MOUMILE K, BERNEDE C, LE BOURGEOIS M, WAERNESSYCKLE S, DESCAMPS P, SERMET-GAUDELUS I, LENOIR G, BERCHE P, TADDEI F

2009 - Pediatr Pulmonol 44(8):820-825

BACKGROUND: Cystic fibrosis patients suffer from recurrent bacterial infections that result in progressive deterioration of their respiratory function. Despite intensive antibiotic treatment, Pseudomonas aeruginosa is the main cause of such infections, with clones progressively developing multiple antibiotic resistance. We determined the relationship between the presence of P. aeruginosa mutator strains and cystic fibrosis clinical characteristics. METHODS: We analyzed 136 strains of P. aeruginosa isolated from the expectorations of 36 CF patients. On all strains, mutation frequencies were determined by the mutation rate to rifampicin, and antibiotic susceptibility was determined by the disk diffusion method. The epidemiological relatedness of these 136 P. aeruginosa strains was studied by pulsed-field gel electrophoresis. The appearance of new antibiotic resistance by sequential analysis of genotypically identical strains was determined. Lung function test results, that is, forced expiratory volume in 1 sec and vital capacity, were also recorded from these patients. RESULTS: We showed that bacteria with an enhanced mutation rate increase the rate of acquisition of new antibiotic resistance threefold and are associated with the deterioration of lung function. CONCLUSIONS: This study demonstrates the effect of mutator bacteria on the efficiency of patient treatment and on their respiratory function. Given the consequence of antibiotic treatment failure and lung deterioration on the prognosis of CF patients, antibiotic treatment strategies may need to be optimized to prevent the emergence of mutator clones. Pediatr Pulmonol. 2009; 44:820-825. (c) 2009 Wiley-Liss, Inc.

Unité(s) : Laboratoire de Microbiologie, Pédiatrie Générale, Pneumologie et Asthmologie Pédiatriques, U571

Prognostic factors for virological response in antiretroviral therapy-naive patients in the MONARK Trial randomized to ritonavir-boosted lopinavir alone

FLANDRE P, DELAUGERRE C, GHOSN J, CHAIX ML, HORBAN A, GIRARD PM, GLADYSZ A, COHEN-CODAR I, VAN PN, TABURET AM, ROUZIOUX C, DELFRAISSY JF

2009 - Antivir. Ther. 14(1):93-97

Background: MONARK is a pilot randomized trial comparing the safety and efficacy of lopinavir/ritonavir (LPV/r) monotherapy to a standard triple-drug regimen as initial therapy. The primary endpoint was virological response (VR) defined as viral load (VL)<400 copies/ml at week 24 and VL<50 copies/ml at week 48. The objective of this study was to determine prognostic factors of VIR in patients receiving LPV/r monotherapy. Methods: Baseline characteristics, including demographics, HIV type-1 (HIV-1) subtype (B versus non-B), early VR up to week 4, LPV trough concentrations and compliance were investigated as prognostic factors for VR in patients receiving LPV/r monotherapy. Logistic regression was used to search for variables significantly associated with the occurrence of VR. Results: VR was achieved in 53 out of 83 patients randomized to the LPV/r arm. The on-treatment analysis, using a multivariate model, indicated that having VL<400 copies/ml at week 4 and harbouring HIV-1 subtype B were independently associated with an increased probability of VR. No difference in early VL reduction was evidenced between patients harbouring B or non-B subtypes. The latter patients had more difficulty in adherence to therapy than the former patients. The intention-to-treat analysis showed similar results. Conclusions: HIV-1 RNA measured at baseline or at week 4 and HIV-1 subtype (B versus non-B) were independent predictive factors of VR in patients starting therapy with LPV/r alone. Although based on a small sample size, results of this study showed that adherence to therapy is lower in patients harbouring non-B subtypes and appears to be a key factor of VR in the context of protease inhibitor monotherapy.

Unité(s) : Laboratoire de Microbiologie

High frequency of X4/DM-tropic viruses in PBMC samples from patients with primary HIV-1 subtype-B infection in 1996-2007: the French ANRS CO06 PRIMO Cohort Study

FRANGE P, GALIMAND J, GOUJARD C, DEVEAU C, GHOSN J, ROUZIOUX C, MEYER L, CHAIX ML

2009 - J Antimicrob Chemother 64(1):135-141

Objectives To estimate the frequency of viruses with X4 or dual-X4/DM tropism from peripheral blood mononuclear cells (PBMCs) of 390 human immunodeficiency virus type 1 (HIV-1) subtype-B patients diagnosed at the time of primary HIV-1 infection (PHI) between 1996 and 2007 and enrolled in the PRIMO Cohort. Methods V3 loop sequences were amplified from HIV-1-DNA and analysed with a combination of five genotypic rules to predict tropism: (i) the '11/25 rule'; (ii) the net charge rule; (iii) the PSSM(X4/DM) algorithm; (iv) the PSSM(SI/NSI) algorithm; and (v) the SVM(Geno2pheno) algorithm. Results A high proportion (62/390, 15.9%) of patients harboured X4/DM-tropic viruses. This prevalence was stable over time: 18.1% before 2003 versus 14.8% since 2003. No difference according to HIV tropism was noted in HIV-RNA levels, CD4 cell count, time between infection and enrolment, and HIV infection risk factor. The frequency of X4/DM-tropic virus was similar among patients infected with a resistant virus (12/62, 19.4%) compared with patients harbouring wild-type strains (50/328, 15.2%). Conclusions This large French epidemiological study evidenced a high proportion of patients (15.9%) harbouring X4/DM-tropic viruses in PBMCs at the time of PHI, suggesting the existence of a cellular X4/DM viral reservoir that could persist for lengthy period of time. Several reports identified that HIV-1 CXCR4 usage was more frequent among patients who developed AIDS and was a powerful predictor of the response to antiretrovirals. Further studies are needed to evaluate the impact of such strains on the outcome of HIV disease, when they are detected at the time of primary infection.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques, EA3620

HIV-1 resistance to first- and second-generation non-nucleoside reverse transcriptase inhibitors

GHOSN J, CHAIX ML, DELAUGERRE C

2009 - AIDS Rev 11(3):165-73

Resistance to the first-generation non-nucleoside reverse transcriptase inhibitors nevirapine and efavirenz is characterized by rapid selection of viruses carrying one or several mutations in the reverse transcriptase gene, which immediately confer high-level resistance as well as cross-resistance to the two drugs. Such mutations have been detected close to the non-nucleoside reverse transcriptase inhibitor binding site and also in the connection domain of HIV reverse transcriptase. They lead to a loss of drug affinity without affecting viral fitness. As a single mutation is enough to confer high-level resistance, transmission of non-nucleoside reverse transcriptase inhibitor-resistant viruses (currently 2-7% of cases) is strongly associated with virologic failure of non-nucleoside reverse transcriptase inhibitor-based first-line regimens. The development of second-generation non-nucleoside reverse transcriptase inhibitors is a major challenge. The most promising compounds, etravirine and rilpivirine, are active on mutant viruses and possess a relatively high genetic barrier for resistance. Data on etravirine resistance in patients already exposed to first-generation non-nucleoside reverse transcriptase inhibitors show that, among 17 mutations in the reverse transcriptase gene, at least three must be present simultaneously in order to diminish etravirine activity. Recent studies of the prevalence of resistance in large databases of patients already exposed to nevirapine and efavirenz show that more than three-quarters of strains will still be sensitive to etravirine in both the southern and northern hemispheres. The first data on rilpivirine resistance are encouraging, but still too preliminary.

Unité(s) : Laboratoire de Microbiologie, EA3620

Rapid selection and archiving of mutation E157Q in HIV-1 DNA during short-term low-level replication on a raltegravir-containing regimen

GHOSN J, MAZET AA, AVETTAND-FENOEL V, PEYTAVIN G, WIRDEN M, DELFRAISSY JF, CHAIX ML

2009 - J Antimicrob Chemother 64(2):433-434

Unité(s) : Laboratoire de Microbiologie, EA3620

Spontaneous control of viral replication during primary HIV infection: when is "HIV controller" status established?

GOUJARD C, CHAIX ML, LAMBOTTE O, DEVEAU C, SINET M, GUERGNON J, COURGNAUD V, ROUZIOUX C, DELFRAISSY JF, VENET A, MEYER L, AGENCE NATIONALE DE RECHERCHE SUR LE SIDA PSG

2009 - Clin Infect Dis 49(6):982-6

Eight patients in the ANRS PRIMO cohort experienced early spontaneous viral control. Viral control was established a median of 6.2 months after primary human immunodeficiency virus type 1 infection and lasted a median of 4.1 years. Seven of the patients initially had detectable viral replication. For 4 patients, viral control was lost during follow-up.

Unité(s) : Laboratoire de Microbiologie, EA3620

Pivotal role of the Francisella tularensis heat-shock sigma factor RpoH

GRALL N, LIVNY J, WALDOR M, BAREL M, CHARBIT A, MEIBOM KL

2009 - Microbiology 155(8):2560-72

Francisella tularensis is a highly infectious pathogen that infects animals and humans to cause the disease tularemia. The primary targets of this bacterium are macrophages, in which it replicates in the cytoplasm after escaping the initial phagosomal compartment. The ability to replicate within macrophages relies on the tightly regulated expression of a series of genes. One of the most commonly used means of coordinating the regulation of multiple genes in bacteria consists of the association of dedicated alternative sigma factors with the core of the RNA polymerase (RNAP). In silico analysis of the F. tularensis LVS genome led us to identify, in addition to the genes encoding the RNAP core (comprising the alpha1, alpha2, beta, beta' and omega subunits), one gene (designated rpoD) encoding the major sigma factor sigma(70), and a unique gene (FTL_0851) encoding a putative alternative sigma factor homologue of the sigma(32) heat-shock family (designated rpoH). Hence, F. tularensis represents one of the minority of bacterial species that possess only one or no alternative sigma factor in addition to the main factor sigma(70). In the present work, we show that FTL_0851 encodes a genuine sigma(32) factor. Transcriptomic analyses of the F. tularensis LVS heat-stress response allowed the identification of a series of orthologues of known heat-shock genes (including those for Hsp40, GroEL, GroES, DnaK, DnaJ, GrpE, ClpB and ClpP) and a number of genes implicated in Francisella virulence. A bioinformatic analysis was used to identify genes preceded by a putative sigma(32)-binding site, revealing both similarities to and differences from RpoH-mediated gene expression in Escherichia coli. Our results suggest that RpoH is an essential protein of F. tularensis, and positively regulates a subset of genes involved in the heat-shock response.

Unité(s) : Laboratoire de Microbiologie, U570

Decreased Risk of Congenital Cytomegalovirus Infection in Children Born to HIV-1-Infected Mothers in the Era of Highly Active Antiretroviral Therapy

GUIBERT G, WARSZAWSKI J, LE CHENADEC J, BLANCHE S, BENMEBAREK Y, MANDELBROT L, TUBIANA R, ROUZIOUX C, LERUEZ-VILLE M, FRENCH PERINATAL C

2009 - Clin Infect Dis 48(11):1516-1525

Background. We evaluated the prevalence of congenital cytomegalovirus (CMV) infection before and after highly active antiretroviral therapy (HAART) availability among neonates born to human immunodeficiency virus type 1 (HIV-1)-infected mothers. We also identified maternal risk factors associated with in utero CMV transmission. Method. Routine screening for congenital CMV infection was performed from 1993 through 2004 in children born to HIV-1-infected mothers included in the French Perinatal Cohort (Enquete Perinatale Francaise). Interpretable tests on urine samples collected within the first 10 days of life were available for 4797 of the 7563 live-born infants. Prevalence was estimated for different time periods. Univariate and multivariate logistic regression analyses were performed to identify factors associated with CMV transmission in the HAART era. Results. Among live-born children, the overall prevalence of CMV infection was 2.3% (95% confidence interval, 1.9%-2.8%). Prevalence was higher among HIV-1-infected neonates (10.3%; 95% confidence interval, 5.6%-17.0%) than among HIV-1-uninfected neonates (2.2%; 95% confidence interval, 1.8%-2.7%; [Formula: see text]). Among HIV-1-uninfected neonates, the prevalence of CMV infection decreased over time, from 3.5% in 1997-1998 to 1.2% in 2003-2004. Delivery period, maternal age, time at antiretroviral treatment initiation, and maternal CD4(+) cell count <200 cells/mm(3) close to delivery were independently associated with CMV infection in logistic regression analysis. The percentage of symptomatic CMV infections was 23.1% among HIV-1-infected newborns and 6.7% among HIV-1-uninfected neonates. Conclusions. The prevalence of congenital CMV infection was high and associated with high morbidity rates among HIV-1-infected neonates. Conversely, the prevalence of CMV infection decreased over time among neonates not infected with HIV-1, reaching levels similar to those observed in the general population, following the introduction and increasing use of HAART for prevention of mother-to-child HIV-1 transmission.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques, EA3620

Didanosine population pharmacokinetics in West African human immunodeficiency virus-infected children administered once-daily tablets in relation to efficacy after one year of treatment

HIRT D, BARDIN C, DIAGBOUGA S, NACRO B, HIEN H, ZOURE E, ROUET F, OUIMINGA A, URIEN S, FOULONGNE V, VAN DE PERRE P, TRELUYER JM, MSELLATI P

2009 - Antimicrob Agents Chemother 53(10):4399-406

Our objective was to study didanosine pharmacokinetics in children after the administration of tablets, the only formulation available in Burkina Faso for which data are missing, and to establish relationships between doses, plasma drug concentrations, and treatment effects (efficacy/toxicity). Didanosine concentrations were measured for 40 children after 2 weeks and for 9 children after 2 to 5 months of treatment with a didanosine-lamivudine-efavirenz combination. A population pharmacokinetic model was developed with NONMEM. The link between the maximal concentration of the drug in plasma (Cmax), the area under the concentration-time curve (AUC), and the decrease in human immunodeficiency virus (HIV) type 1 RNA levels after 12 months of treatment was evaluated. The threshold AUC that improved efficacy was determined by the use of a Wilcoxon test for HIV RNA, and an optimized dosing schedule was simulated. Didanosine pharmacokinetics was best described by a one-compartment model with first-order absorption and elimination. The apparent clearance and volume of distribution were higher for tablets, probably due to a lower bioavailability with tablets than with pediatric powder. The decrease in the viral load after 12 months of treatment was significantly correlated with the didanosine AUC and Cmax (P < or = 0.02) during the first weeks of treatment. An AUC of >0.60 mg/liter x h was significantly linked to a greater decrease in the viral load (a decrease of 3 log10 versus 2.4 log10 copies/ml; P = 0.03) than that with a lower AUC. A didanosine dose of 360 mg/m2 administered as tablets should be a more appropriate dose than 240 mg/m2 to improve efficacy for these children. However, data on adverse events with this dosage are missing.

Unité(s) : Laboratoire de Microbiologie

Is the recommended dose of efavirenz optimal in young West African human immunodeficiency virus-infected children?

HIRT D, URIEN S, OLIVIER M, PEYRIERE H, NACRO B, DIAGBOUGA S, ZOURE E, ROUET F, HIEN H, MSELLATI P, VAN DE PERRE P, TRELUYER JM

2009 - Antimicrob Agents Chemother 53(10):4407-13

We aimed in this study to describe efavirenz concentration-time courses in treatment-naive children after once-daily administration to study the effects of age and body weight on efavirenz pharmacokinetics and to test relationships between doses, plasma concentrations, and efficacy. For this purpose, efavirenz concentrations in 48 children were measured after 2 weeks of didanosine-lamivudine-efavirenz treatment, and samples were available for 9/48 children between months 2 and 5 of treatment. Efavirenz concentrations in 200 plasma specimens were measured using a validated high-performance liquid chromatography method. A population pharmacokinetic model was developed with NONMEM. The influence of individual characteristics was tested using a likelihood ratio test. The estimated minimal and maximal concentrations of efavirenz in plasma (Cmin and Cmax, respectively) and the area under the concentration-time curve (AUC) were correlated to the decrease in human immunodeficiency virus type 1 RNA levels after 3 months of treatment. The threshold Cmin (and AUC) that improved efficacy was determined. The target minimal concentration of 4 mg/liter was considered for toxicity. An optimized dosing schedule that would place the highest percentage of children in the interval of effective and nontoxic concentrations was simulated. The pharmacokinetics of efavirenz was best described by a one-compartment model with first-order absorption and elimination. The mean apparent clearance and volume of distribution for efavirenz were 0.211 liter/h/kg and 4.48 liters/kg, respectively. Clearance decreased significantly with age. When the recommended doses were given to 46 of the 48 children, 19% (44% of children weighing less than 15 kg) had C(min)s below 1 mg/liter. A significantly higher percentage of children with C(min)s of >1.1 mg/liter or AUCs of >51 mg/liter x h than of children with lower values had viral load decreases greater than 2 log10 copies/ml after 3 months of treatment. Therefore, to optimize the percentage of children with C(min)s between 1.1 and 4 mg/liter, children should receive the following once-daily efavirenz doses: 25 mg/kg of body weight from 2 to 6 years, 15 mg/kg from 6 to 10 years, and 10 mg/kg from 10 to 15 years. These assumptions should be prospectively confirmed.

Unité(s) : Laboratoire de Microbiologie

A two-component system is required for colonization of host cells by meningococcus

JAMET A, ROUSSEAU C, MONFORT JB, FRAPY E, NASSIF X, MARTIN P

2009 - Microbiology 155(7):2288-95

In order to adapt to changing environments, bacteria have evolved two-component systems (TCSs) that are able to sense and respond to environmental stimuli. The signal perception relies on a sensor protein whose activation allows rapid adaptation through transcriptional regulation achieved by the regulatory protein. The ability to adhere to and grow on the surface of human host cells is an absolute requirement for many pathogens, including Neisseria meningitidis, in order to colonize new hosts and to disseminate inside their host. Among the four TCSs encoded in the meningococcus genome, only the PhoQ (MisS)/PhoP (MisR) system has been shown to constitute a functional signal transduction circuit. To investigate the involvement of this TCS in the adaptation process requisite for host cell colonization, we have tested the ability to grow on host cells of a mutant inactivated for the sensor of the TCS. Our results demonstrate the involvement of the TCS in the adaptation of the meningococcus to growth on host cells. We show that the expression of the PhoQ (MisS)/PhoP (MisR) TCS is cell-contact controlled. Furthermore, this TCS controls the regulation of a group of genes, the REP2 regulon, previously shown to be cell-contact regulated and to encode functions crucial for the adaptation of the bacterium to host cell colonization. Thus, we provide evidence that one of the four TCSs existing in N. meningitidis contributes to the adaptation of the pathogen to growth on host cells.

Unité(s) : Laboratoire de Microbiologie, U570

Low prevalence of drug resistance transmitted virus in HIV Type 1-infected ARV-naive patients in Cambodia

JANIN N, SOPHEAK N, REGIS MP, OLIVIER M, SIM KL, FREDERIC A, MARTINE P, MARIE-LAURE C, AHIDJO A, ERIC N

2009 - AIDS Res Hum Retroviruses 25(5):543-5

Abstract Between November 2006 and June 2007, HIV-1 reverse transcriptase (RT) and protease (PR) genes of 67 ARV-naive Cambodian patients were amplified and sequenced. At inclusion, the median age and duration of HIV infection were 28 and 1.1 years, respectively. The median CD4 and HIV-1 RNA were 611 cells/ml [IQR: 525-759] and 4.0 log(10) copies/ml [IQR: 3.4-4.6]. Among 67 HIV-1 strains, 95.5% were CRF 01_AE viruses (n = 64) whereas three clustered with subtype B. RT analysis indicated that only 1 patient out of 67, presenting K103N and M184V mutations, was resistant to NVP/EFV and 3TC/FTC. No primary resistance to protease inhibitors was detected in 59 amplified protease genes. The 1.49% (IC 95%: 0.04-8.04%) prevalence of transmitted drug-resistant strains in drug-naive patients was low in our study. Surveys of drug-resistant transmitted viruses should be regularly performed regarding the increasing access to HAART in Cambodia.

Unité(s) : Laboratoire de Microbiologie, EA3620

Is granulomatous mastitis a localized form of hidradenitis suppurativa?

JOIN-LAMBERT O, FRAITAG S, RIBADEAU-DUMAS F, LEGUERN AS, BEHILLIL S, DEL CASTILLO FJ, CONSIGNY PH, AUQUIER F, EB F, SEVESTRE H, LORTHOLARY O, NASSIF X, NASSIF A

2009 - Eur J Dermatol 19(5):513-514

Unité(s) : Laboratoire de Microbiologie, Anatomie Pathologique, Maladies Infectieuses

Microsporidiosis in solid organ transplant recipients: two Enterocytozoon bieneusi cases and review

LANTERNIER F, BOUTBOUL D, MENOTTI J, CHANDESRIS MO, SARFATI C, MAMZER-BRUNEEL MF, CALMUS Y, MECHAI F, VIARD JP, LECUIT M, BOUGNOUX ME, LORTHOLARY O

2009 - Transpl. Infect. Dis. 11(1):83-88

Microsporidiosis first came to prominence as an opportunistic infection in patients with acquired immunodeficiency syndrome. Microsporidia are now emerging pathogens responsible for severe diarrhea during solid organ transplantation. Two main clinical entities can be identified: infection by Enterocytozoon bieneusi, causing diarrhea with limited treatment options; and infection by Encephalitozoon intestinalis, which may disseminate and usually responds to albendazole treatment. We describe here 2 cases of microsporidiosis caused by E. bieneusi in a renal and a liver transplant recipient, respectively, in whom complete clinical efficacy of a short course of fumagillin therapy was obtained. Long-term microbiological eradication was assessed using classical methods and monitored using a real-time quantitative polymerase chain reaction-based method. Both patients experienced drug-induced thrombocytopenia, which resolved after withdrawal of the treatment. We also review the 18 other previously reported cases of microsporidiosis in transplant recipients. In case of persistent diarrhea in solid organ transplant patients, microsporidiosis should be considered. Based on the present experience, treating E. bieneusi infection with 7 days of fumagillin therapy is adequate to eradicate E. bieneusi in this context.

Unité(s) : Maladies Infectieuses, Laboratoire de Microbiologie, Transplantation Adulte

Therapeutic drug monitoring of posaconazole: a monocentric study in 54 adults

LEBEAUX D, LANTERNIER F, ELIE C, SUAREZ F, BUZYN A, VIARD JP, BOUGNOUX ME, LECUIT M, JULLIEN V, LORTHOLARY O

2009 - Antimicrob Agents Chemother 53(12):5224-29

Posaconazole is a potent broad-spectrum triazole antifungal for which little is known about the prevalence and risk factors for low plasma concentration. We retrospectively reviewed all adult patients who underwent measurement of posaconazole plasma concentration (PPC) after at least 5 days of treatment between April 2006 and July 2008 at the Hopital Necker Enfants Malades. Low PPC was defined as a concentration lower than 500 ng/mL. Fifty-four patients were included: 36 receiving prophylactic (200 mg t.i.d.) and 18 curative posaconazole therapy (400 mg b.i.d.). Prevalence of low PPC was 44% (16/36) in the prophylaxis group and 22% (4/18) in the curative treatment group. In the prophylaxis group, low PPC tended to be more frequent in case of digestive disease (62.5% vs. 30%, p=0.051) and was significantly more frequent in patients with diarrhea (71.4% vs. 27%, p=0.009) or mucositis (100% vs. 33%, p=0.004). In the curative treatment group, low PPC was significantly more frequent in case of diarrhea (75% vs. 7%, p=0.018). In the prophylaxis group, the only 2 patients who subsequently developed invasive fungal infection exhibited a low PPC. The only adverse event was hepatotoxicity in 2/54 patients (3.7%), not related to high plasma concentrations. In conclusion, low PPC is common, significantly more frequent in case of diarrhea or mucositis and potentially associated with subsequent invasive fungal infection. Posaconazole therapeutic drug monitoring is therefore mandatory in immunosuppressed adults, at least in those with gastrointestinal disorder.

Unité(s) : Laboratoire de Microbiologie, Biostatistique, Hématologie Adulte, Maladies Infectieuses

Antiretroviral therapy initiation during primary HIV infection enhances both CD127 expression and the proliferative capacity of HIV-specific CD8+ T cells

LECUROUX C, GIRAULT I, BOUTBOUL F, URRUTIA A, GOUJARD C, MEYER L, LAMBOTTE O, CHAIX ML, MARTINEZ V, AUTRAN B, SINET M, VENET A, ANRS PRIMO COHORT AHICSG, COHORT AA, GROUP AHS

2009 - AIDS 23(13):1649-58

OBJECTIVES: HIV-specific CD8+ T cells from patients with primary HIV infection (PHI) and after antiretroviral therapy initiation were evaluated for CD127 expression and proliferative capacity and were compared with cells from chronically-infected patients, including long-term nonprogressors and HIV controllers. METHODS: We studied 30 patients with PHI (from the Agence Nationale de Recherche sur le SIDA Primo-infection Cohort) and 33 patients with chronic HIV infection (including nonprogressor patients from the Agence Nationale de Recherche sur le SIDA ALT Cohort and the Agence Nationale de Recherche sur le SIDA HIV Controllers Study Group). HIV-specific CD8+ T cells were identified by costaining with HIV human leukocyte antigen class I pentamers. CD127 expression was assessed by flow cytometry and cell proliferation by carboxyfluorescein succinimidyl ester labeling. RESULTS: During PHI, most HIV-specific CD8+ T cells coexpressed CD27 and CD45RO, were highly activated, and showed weak Bcl-2 expression. Their CD127 expression was very low and correlated negatively both with HIV RNA and DNA levels and with expression of the activation marker CD38. CD127 expression correlated positively with CD4 cell count, Bcl-2 expression and proliferative capacity. Strong CD127 expression was observed in the two groups of chronically-infected nonprogressors. CD127 expression on HIV-specific CD8+ T cells increased in early-treated PHI patients, reaching levels similar to those observed in nonprogressors. In parallel, these cells acquired strong proliferative capacity. No change in CD127 expression or proliferative potential was observed in untreated patients. CONCLUSION: Early antiretroviral therapy initiation enhances CD127 expression on HIV-specific CD8+ T cells, reaching levels similar to those observed in aviremic nonprogressors, and restores their proliferative capacity.

Unité(s) : Laboratoire de Microbiologie

Influence of the HCV subtype on the virological response to pegylated interferon and ribavirin therapy

LEGRAND-ABRAVANEL F, COLSON P, LEGUILLOU-GUILLEMETTE H, ALRIC L, RAVAUX I, LUNEL-FABIANI F, BOUVIERS-ALIAS M, TRIMOULET P, CHAIX ML, HEZODE C, FOUCHER J, FONTAINE H, ROQUE-AFONSO AM, GASSIN M, SCHVOERER E, GAUDY C, ROCHE B, DOFFOEL M, D'ALTEROCHE L, VALLET S, BAAZIA Y, POZZETTO B, THIBAULT V, NOUSBAUM JB, ROULOT D, COPPERE H, POINARD T, PAYAN C, IZOPET J

2009 - J Med Virol 81(12):2029-35

The hepatitis C virus genotype is considered to be the most important baseline predictor of a sustained virological response in patients with chronic hepatitis C treated with pegylated interferon and ribavirin. The influence of the subtype on the sustained virological response was investigated in patients infected with genotypes 1, 4, 5, or 6. This study was done on 597 patients with chronic hepatitis C who were given pegylated interferon and ribavirin for 48 weeks. The overall rate of sustained virological response in the 597 patients was 37.8%. Univariate analysis indicated that the sustained virological response of patients infected with subtype 1b (39%) tended to be higher than that of patients infected with subtype 1a (30.6%; P = 0.06) and it was similar to those patients infected with subtypes 4a (51.3%; P = 0.12) or 4d (51.7%; P = 0.16). Multivariate analysis indicated that five factors were independently associated with sustained virological response: the age (OR 0.97; 95% CI = 0.95-0.99), absence of cirrhosis (OR: 2.92; 95% CI = 1.7-5.0; P < 0.01), absence of HIV co-infection (OR: 2.08; 95% CI = 1.2-3.5; P < 0.01), low baseline plasma HCV RNA concentration (OR: 1.74; 95% CI = 1.2-2.6; P < 0.01), and the subtype 1b (OR: 1.61; 95% CI = 1.0-2.5; P = 0.04) or subtypes 4a and 4d (OR: 2.03; 95% CI = 1.1-3.8; P = 0.03). In conclusion, among difficult-to-treat genotypes, the subtype 1a is associated with a lower response to anti-HCV therapy than subtypes 1b, 4a, and 4d.

Unité(s) : Laboratoire de Microbiologie

[Retrospective diagnosis of congenital CMV infection in DBS from Guthrie cards: French experience.]

LERUEZ-VILLE M, VAULOUP-FELLOUS C, COUDERC S, PARAT S, OUCHERIF S, CASTEL C, MAGNY JF

2009 - Arch Pediatr 16(11):1503-06

Systematic screening for cytomegalovirus congenital infection is not performed in France. For children with hearing loss or other neurological CMV compatible symptoms, retrospective diagnosis is possible by PCR detection of CMV DNA in dried blood spot of neonatal Guthrie cards. We report here the results obtained with this technique in the French national reference laboratory for cytomegalovirus.

Unité(s) : Laboratoire de Microbiologie, Obstétrique

Enhanced T cell recovery in HIV-1-infected adults through IL-7 treatment

LEVY Y, LACABARATZ C, WEISS L, VIARD JP, GOUJARD C, LELIEVRE JD, BOUE F, MOLINA JM, ROUZIOUX C, AVETTAND-FENOEL V, CROUGHS T, BEQ S, THIEBAUT R, CHENE G, MORRE M, DELFRAISSY JF

2009 - J Clin Invest 119(4):997-1007

HIV infection results in CD4+ T cell deficiency, but efficient combination antiretroviral therapy (c-ART) restores T cells and decreases morbidity and mortality. However, immune restoration by c-ART remains variable, and prolonged T cell deficiency remains in a substantial proportion of patients. In a prospective open-label phase I/IIa trial, we evaluated the safety and efficacy of administration of the T cell regulator IL-7. The trial included 13 c-ART-treated HIV-infected patients whose CD4+ cell counts were between 100 and 400 cells/microl and plasma HIV RNA levels were less than 50 copies/ml. Patients received a total of 8 subcutaneous injections of 2 different doses of recombinant human IL-7 (rhIL-7; 3 or 10 microg/kg) 3 times per week over a 16-day period. rhIL-7 was well tolerated and induced a sustained increase of naive and central memory CD4+ and CD8+ T cells. In the highest dose group, 4 patients experienced transient increases in viral replication. However, functional assays showed that the expanded T cells responded to HIV antigen by producing IFN-gamma and/or IL-2. In conclusion, in lymphopenic HIV-infected patients, rhIL-7 therapy induced substantial functional and quantitative changes in T cells for 48 weeks. Therefore, patients may benefit from intermittent therapy with IL-7 in combination with c-ART.

Unité(s) : Maladies Infectieuses, Laboratoire de Microbiologie

Early Control of HIV-1 Infection in Long-Term Nonprogressors Followed Since Diagnosis in the ANRS SEROCO/HEMOCO Cohort

MADEC Y, BOQFASSA F, AVETTAND-FENOEL V, HENDOU S, MELARD A, BOUCHERIT S, SURZYN J, MEYER L, ROUZIOUX C

2009 - JAIDS 50(1):19-26

Background: To clarify early correlates and natural history of HIV long-term nonprogressors (LTNPs) since HIV diagnosis. Methods: Patients enrolled in the French ANRS SEROCO/HEMOCO cohort with CD4 count >500 cells/mm(3) at HIV diagnosis. LTNP status was defined as being asymptomatic, antiretroviral free, and with CD4 cell count >500 cells/mm(3) for >8 years after HIV diagnosis. In LTNPs, we modeled the biological markers' progression through a joint model. Factors associated with loss of LTNP status were identified through a Cox model. Results: Sixty (9%) of 664 patients were identified as LTNPs during follow-up. At enrollment, HIV RNA was <= 2.6 log copies/mL in 24% of LTNPs and HIV DNA was <= 1.85 log copies/10(6) peripheral blood mononuclear cells (PBMCs) in 31% vs. 3% and 8% in others. In LTNPs. HIV RNA and HIV DNA levels increased by 0.04 log copies/mL per year and 0.07 log copies/10(6) PBMCs per year during the: first 8 years after diagnosis. LTNP status was lost in 36 subjects;, baseline HIV DNA >1.85 log copies/10(6) PBMCs and high HIV DNA increase were associated with an increased risk of losing LTNP status adjusted hazard ratio: 2.8 (1.2-6.8) and 2.2 (1.0-4.8), respectively). Conclusions: LTNP status is established in the first years of HIV infection, low HIV DNA level at enrollment and slow increase of HIV DNA being associated with maintained LTNP status.

Unité(s) : Laboratoire de Microbiologie, EA3620

Amniocentesis and mother-to-child human immunodeficiency virus transmission in the Agence Nationale de Recherches sur le SIDA et les Hepatites Virales French Perinatal Cohort

MANDELBROT L, JASSERON C, EKOUKOU D, BATALLAN A, BONGAIN A, PANNIER E, BLANCHE S, TUBIANA R, ROUZIOUX C, WARSZAWSKI J, COHORT AFP

2009 - Am J Obstet Gynecol 200(2):160 e1-9

OBJECTIVE: The objective of the study was to investigate whether performing an amniocentesis increased mother-to-child transmission of human immunodeficiency virus (HIV)-1 (MTCT). STUDY DESIGN: We studied HIV -1 infected mothers and their children enrolled in the multicenter French Perinatal HIV Cohort from 1985 to 2006. RESULTS: One hundred sixty-six amniocenteses were performed among 9302 singleton pregnancies, the proportion increasing from 1.0% before 2001 to 4.7% in 2005-2006. Use of highly active antiretroviral therapy (HAART) was more frequent in the amniocentesis group (58.4% vs 33.2%). MTCT tended to be higher in the amniocentesis group, among mothers who received no antiretroviral agents (25.0%; 3/12 vs 16.2%; 343/2113; P = .41) as well as among mothers receiving zidovudine monotherapy or a double-nucleoside reverse transcriptase inhibitor combination (6.1%; 3/49 vs 3.3%; 117/3556; P = .22), but the difference was not significant. Among 81 mothers receiving HAART, there was no case of MTCT. CONCLUSION: Our results suggest that amniocentesis is not a major risk factor for mother-to-child transmission in mothers treated with effective antiretroviral therapy.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie

No association between human herpesvirus 6 reactivation and cryptococcosis in human immunodeficiency virus-infected patients

MICOL R, BUCHY P, GALIMAND J, VEASNA D, FERRADINI L, BALKAN S, GUERIN PJ, MARTIN PR, FONTANET A, LORTHOLARY O, ROUZIOUX C, LERUEZ-VILLE M

2009 - J. Med. Microbiol. 58(Pt.2):276-277

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses

Prevalence, risk factors, and impact on outcome of cytomegalovirus replication in serum of Cambodian HIV-infected patients (2004-2007)

MICOL R, BUCHY P, GUERRIER G, DUONG V, FERRADINI L, DOUSSET JP, GUERIN PJ, BALKAN S, GALIMAND J, CHANROEUN H, LORTHOLARY O, ROUZIOUX C, FONTANET A, LERUEZ-VILLE M

2009 - JAIDS 51(4):486-91

BACKGROUND: In developing countries, the study of cytomegalovirus (CMV) coinfection in HIV-infected patients remains neglected. Quantitative CMV polymerase chain reaction (PCR) is the gold standard diagnostic tool for analyzing serum CMV replication and for predicting CMV disease. We estimated the prevalence of replicating CMV in sera of newly diagnosed HIV-infected Cambodian patients and examined its impact on mortality. METHODS: This cohort study was based on 2 highly active antiretroviral therapy treatment programs in Cambodia between 2004 and 2007. Quantitative CMV PCR was performed on baseline serum samples of 377 HIV-infected patients. RESULTS: The prevalence of serum CMV DNA was 55.2% (150 of 272) in patients with CD4 count <100/mm. In multivariate analysis, hemoglobin <9 g/dL, CD4 count <100/mm, and Karnofsky index <50 were independently associated with positive serum CMV DNA at baseline. During a 3-year follow-up period, CMV viral load >or=3.1 log10 copies per milliliter was significantly associated with death independently of CD4 count, other opportunistic infections, and highly active antiretroviral therapy. CONCLUSIONS: As in industrialized countries, serum CMV replication is highly prevalent among HIV-infected Cambodian patients and is associated with increased mortality. This underscores the importance of diagnostic CMV infection by PCR in sera of HIV-infected patients with CD4 count <100/mm and treating this opportunistic infection to reduce its associated mortality.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses

Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress

MIKATY G, SOYER M, MAIREY E, HENRY N, DYER D, FOREST KT, MORAND P, GUADAGNINI S, PREVOST MC, NASSIF X, DUMENIL G

2009 - PLoS Pathog 5(2):e1000314

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow-generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.

Unité(s) : U570, Laboratoire de Microbiologie

A revolution in the identification of pathogens in clinical laboratories

NASSIF X

2009 - Clin Infect Dis 49(4):552-3

Unité(s) : Laboratoire de Microbiologie, U570

Tracing the HIV-1 subtype B mobility in Europe: a phylogeographic approach

PARASKEVIS D, PYBUS O, MAGIORKINIS G, HATZAKIS A, WENSING AM, VAN DE VIJVER DA, ALBERT J, ANGARANO G, ASJO B, BALOTTA C, BOERI E, CAMACHO R, CHAIX ML, COUGHLAN S, COSTAGLIOLA D, DE LUCA A, DE MENDOZA C, DERDELINCKX I, GROSSMAN Z, HAMOUDA O, HOEPELMAN I, HORBAN A, KORN K, KUCHERER C, LEITNER T, LOVEDAY C, MACRAE E, MALJKOVIC-BERRY I, MEYER L, NIELSEN C, DE COUL ELMO, ORMAASEN V, PERRIN L, PUCHHAMMER-STOCKL E, RUIZ L, SALMINEN MO, SCHMIT JC, SCHUURMAN R, SORIANO V, STANCZAK J, STANOJEVIC M, STRUCK D, VAN LAETHEM K, VIOLIN M, YERLY S, ZAZZI M, BOUCHER CA, VANDAMME AM, PROGRAMME S

2009 - Retrovirology 6(.):49

BACKGROUND: The prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced. RESULTS: In the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred. CONCLUSION: Subtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.

Unité(s) : Laboratoire de Microbiologie, EA3620

Actinomyces in chronic granulomatous disease: an emerging and unanticipated pathogen

REICHENBACH J, LOPATIN U, MAHLAOUI N, BEOVIC B, SILER U, ZBINDEN R, SEGER RA, GALMICHE L, BROUSSE N, KAYAL S, GUNGOR T, BLANCHE S, HOLLAND SM

2009 - Clin Infect Dis 49(11):1703-10

BACKGROUND: Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. METHODS: Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/or a complement fixation test in 10 patients with CGD. RESULTS: All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. CONCLUSIONS: Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.

Unité(s) : Laboratoire de Microbiologie, Anatomie Pathologique, Immunologie-Hématologie Pédiatriques

Relationship of CD4+ T-cell counts and plasma HIV-1 RNA levels with serological HBeAg/anti-HBe patterns obtained in West-African HBV-HIV-1-co-infected children

ROUET F, CHAIX ML, KPOZEHOUEN A, INWOLEY A, ANAKY MF, FASSINOU P, ROUZIOUX C, BLANCHE S, MSELLATI P

2009 - J Trop Pediatr 55(6):409-12

HBeAg/anti-HBe and hepatitis B virus (HBV) DNA from 34 HIV-1-infected children from Ivory Coast with chronic hepatitis B (CHB) were longitudinally analyzed according to CD4 and HIV-1 RNA. The mean CD4% value was significantly (p = 0.03) lower in 59 (52.7%) samples showing a usual CHB (HBeAg-positive/anti-HBe-negative and HBV DNA-positive), as compared with 30 (26.8%) HBeAg-positive/anti-HBe-positive and HBV DNA-positive and 23 (20.5%) HBeAg-negative/anti-HBe-positive and HBV DNA-negative (15.1% vs. 18.5% and 20.0%). The mean HIV-1 RNA concentrations were significantly (p = 0.01) higher in specimens HBV DNA-positive (4.47 and 4.30 log(10)/ml, respectively) vs. HBV DNA-negative (3.43 log(10)/ml). HIV-1 has a significant impact on CHB acquired in childhood.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques, EA3620

Heterogeneity in HIV suppression by CD8 T cells from HIV controllers: association with Gag-specific CD8 T cell responses

SAEZ-CIRION A, SINET M, SHIN SY, URRUTIA A, VERSMISSE P, LACABARATZ C, BOUFASSA F, AVETTAND-FENOEL V, ROUZIOUX C, DELFRAISSY JF, BARRE-SINOUSSI F, LAMBOTTE O, VENET A, PANCINO G, GROUP AEHCS

2009 - J Immunol 182(12):7828-37

"HIV controllers" (HICs) are rare individuals in whom HIV-1 plasma viral load remains undetectable without antiretroviral treatment. This spontaneous viral control in HICs is usually associated to strong functional HIV-specific CD8(+) T cell responses. Accordingly, we have recently shown that CD8(+) T cells from HICs strongly suppress ex vivo HIV-1 infection of autologous CD4(+) T cells, suggesting a crucial role of this response in vivo. Knowledge of the mechanisms underlying the CD8(+) T cell antiviral activity might help to develop effective T cell-based vaccines. In the present work, we further characterized the HIV-suppressive capacity of CD8(+) T cells in 19 HICs. CD8(+) T cells from 14 of the 19 HICs showed strong HIV-suppressive capacity ex vivo. This capacity was stable over time and was partially effective even on other primate lentiviruses. HIV-suppressive capacity of CD8(+) T cells correlated strongly with the frequency of HIV-specific CD8(+) T cells, and in particular of Gag-specific CD8(+) T cells. We also identified five HICs who had weak HIV-suppressive CD8(+) T cell capacities and HIV-specific CD8(+) T cell responses. Among these five HICs, at least three had highly in vitro replicative viruses, suggesting that the control of viremia in these patients is not due to replication-defective viruses. These results, on the one hand, suggest the importance of Gag responses in the antiviral potency of CD8(+) T cells from HICs and, on the other hand, propose that other host mechanisms may contribute to restraining HIV infection in HICs.

Unité(s) : Laboratoire de Microbiologie

Cyst infections in patients with autosomal dominant polycystic kidney disease

SALLEE M, RAFAT C, ZAHAR JR, PAULMIER B, GRUNFELD JP, KNEBELMANN B, FAKHOURI F

2009 - Clin J Am Soc Nephrol 4(7):1183-9

BACKGROUND AND OBJECTIVES: Cyst infection is a complex diagnostic and therapeutic issue in patients with autosomal dominant polycystic kidney disease (ADPKD); however, published data regarding the diagnosis and the management of cyst infections in patients with ADPKD are sparse. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A retrospective study was conducted in a referral center for patients with ADPKD in Paris, France. We identified using a computerized database all patients who had ADPKD and were admitted in the nephrology department of Hopital Necker between January 1998 and August 2008 with likely or definite renal and/or hepatic cyst infection. Medical files of all included patients were reviewed. RESULTS: Among 389 identified patients with ADPKD, 33 (8.4%) had 41 episodes of cyst infection, including eight definite and 33 likely cases. The incidence of cyst infections in patients with ADPKD was 0.01 episode per patient per year. Microbiological documentation was available for 31 episodes (75%), Escherichia coli accounting for 74% of all retrieved bacterial strains. Positron emission tomography scan proved superior to ultrasound, Computed tomography scan, and magnetic resonance imaging for the detection of infected cysts. Clinical efficacy of initial antibiotic treatment was noted in 71% of episodes. Antibiotic treatment modification was more frequently required for patients who were receiving initial monotherapy compared with those who were receiving bitherapy. Large (diameter >5 cm) infected cysts frequently required drainage. CONCLUSIONS: Positron emission tomography scan will probably make the diagnosis of cyst infections easier and more accurate. Antibiotic association, including a fluoroquinolone, and the drainage of large infected cysts remain the main treatment for cyst infections.

Unité(s) : Laboratoire de Microbiologie, Néphrologie Adulte

Raltegravir, etravirine and r-darunavir combination in adolescents with multidrug-resistant virus

THURET I, CHAIX ML, TAMALET C, RELIQUET V, FIRTION G, TRICOIRE J, RABAUD C, FRANGE P, AUMAITRE H, BLANCHE S

2009 - AIDS 23(17):2364-6

Twelve heavily pretreated, perinatally infected adolescents in virological failure were treated with a combination of raltegravir, r-darunavir and etravirine, as part of an expanded access program in France. After a 12-month median follow-up, viral load was <400 copies/ml in 11 (<50 in six). No grade > 2 side effects were recorded. Additional data and marketing authorizations are awaited, but preliminary results in adolescents with extensive multidrug resistant virus are encouraging.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques, EA3620

Chlorhexidine-impregnated sponges and less frequent dressing changes for prevention of catheter-related infections in critically ill adults: a randomized controlled trial

TIMSIT JF, SCHWEBEL C, BOUADMA L, GEFFROY A, GARROUSTE-ORGEAS M, PEASE S, HERAULT MC, HAOUACHE H, CALVINO-GUNTHER S, GESTIN B, ARMAND-LEFEVRE L, LEFLON V, CHAPLAIN C, BENALI A, FRANCAIS A, ADRIE C, ZAHAR JR, THUONG M, ARRAULT X, CROIZE J, LUCET JC, DRESSING STUDY G

2009 - JAMA 301(12):1231-41

CONTEXT: Use of a chlorhexidine gluconate-impregnated sponge (CHGIS) in intravascular catheter dressings may reduce catheter-related infections (CRIs). Changing catheter dressings every 3 days may be more frequent than necessary. OBJECTIVE: To assess superiority of CHGIS dressings regarding the rate of major CRIs (clinical sepsis with or without bloodstream infection) and noninferiority (less than 3% colonization-rate increase) of 7-day vs 3-day dressing changes. DESIGN, SETTING, AND PATIENTS: Assessor-blind, 2 x 2 factorial, randomized controlled trial conducted from December 2006 through June 2008 and recruiting patients from 7 intensive care units in 3 university and 2 general hospitals in France. Patients were adults (>18 years) expected to require an arterial catheter, central-vein catheter, or both inserted for 48 hours or longer. INTERVENTIONS: Use of CHGIS vs standard dressings (controls). Scheduled change of unsoiled adherent dressings every 3 vs every 7 days, with immediate change of any soiled or leaking dressings. MAIN OUTCOME MEASURES: Major CRIs for comparison of CHGIS vs control dressings; colonization rate for comparison of 3- vs 7-day dressing changes. RESULTS: Of 2095 eligible patients, 1636 (3778 catheters, 28,931 catheter-days) could be evaluated. The median duration of catheter insertion was 6 (interquartile range [IQR], 4-10) days. There was no interaction between the interventions. Use of CHGIS dressings decreased the rates of major CRIs (10/1953 [0.5%], 0.6 per 1000 catheter-days vs 19/1825 [1.1%], 1.4 per 1000 catheter-days; hazard ratio [HR], 0.39 [95% confidence interval {CI}, 0.17-0.93]; P = .03) and catheter-related bloodstream infections (6/1953 catheters, 0.40 per 1000 catheter-days vs 17/1825 catheters, 1.3 per 1000 catheter-days; HR, 0.24 [95% CI, 0.09-0.65]). Use of CHGIS dressings was not associated with greater resistance of bacteria in skin samples at catheter removal. Severe CHGIS-associated contact dermatitis occurred in 8 patients (5.3 per 1000 catheters). Use of CHGIS dressings prevented 1 major CRI per 117 catheters. Catheter colonization rates were 142 of 1657 catheters (7.8%) in the 3-day group (10.4 per 1000 catheter-days) and 168 of 1828 catheters (8.6%) in the 7-day group (11.0 per 1000 catheter-days), a mean absolute difference of 0.8% (95% CI, -1.78% to 2.15%) (HR, 0.99; 95% CI, 0.77-1.28), indicating noninferiority of 7-day changes. The median number of dressing changes per catheter was 4 (IQR, 3-6) in the 3-day group and 3 (IQR, 2-5) in the 7-day group (P < .001). CONCLUSIONS: Use of CHGIS dressings with intravascular catheters in the intensive care unit reduced risk of infection even when background infection rates were low. Reducing the frequency of changing unsoiled adherent dressings from every 3 days to every 7 days modestly reduces the total number of dressing changes and appears safe. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00417235.

Unité(s) : Laboratoire de Microbiologie

No evidence of a change in HIV-1 virulence since 1996 in France

TROUDE P, CHAIX ML, TRAN L, DEVEAU C, SENG R, DELFRAISSY JF, ROUZIOUX C, GOUJARD C, MEYER L, COHORT AP

2009 - AIDS 23(10):1261-7

OBJECTIVE: We investigated temporal trends in the CD4 cell count and in plasma HIV RNA and total HIV DNA levels measured at the time of primary HIV infection, as proxies for HIV-1 virulence, taking changes in patient characteristics into account. DESIGN: We studied 903 patients enrolled during primary HIV infection in the French multicenter ANRS PRIMO cohort from 1996 to 2007. METHODS: Associations between the year of primary HIV infection and the values of the three markers were tested with regression models. The year of primary HIV infection was first introduced as a restricted cubic splines function in a regression model in order to explore the shape of the associations, and then as a continuous/categorical variable. The following confounders were considered in multiple regression analysis: time since infection and age (introduced as restricted cubic spline functions), sex, place of birth (Africa vs. others), symptomatic primary HIV infection, smoking, and virus-related factors (subtype B vs. non-B, and drug resistance mutations). RESULTS: Multivariate analysis showed no temporal trends in the CD4 cell count (square-root) or in HIV-1 RNA and DNA levels (log10) measured at the time of primary HIV infection. We observed the well described associations between the prognostic markers and the time since infection, sex, symptomatic primary HIV infection, and smoking. CONCLUSION: The CD4 cell count and HIV RNA and DNA levels measured at the time of primary HIV-1 infection remained stable across 12 consecutive years (1996-2007) in the ANRS PRIMO cohort, suggesting no major change in virulence, after taking into account changes in patient characteristics.

Unité(s) : Laboratoire de Microbiologie, EA3620

Evidence of a large, international network of HCV transmission in HIV-positive men who have sex with men

VAN DE LAAR T, PYBUS O, BRUISTEN S, BROWN D, NELSON M, BHAGANI S, VOGEL M, BAUMGARTEN A, CHAIX ML, FISHER M, GOTZ H, MATTHEWS GV, NEIFER S, WHITE P, RAWLINSON W, POL S, ROCKSTROH J, COUTINHO R, DORE GJ, DUSHEIKO GM, DANTA M

2009 - Gastroenterology 136(5):1609-17

BACKGROUND & AIMS: Since 2000, there has been a marked rise in acute hepatitis C virus (HCV) in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM). We conducted an international phylogenetic study to investigate the existence of an HCV transmission network among MSM. METHODS: HIV-positive MSM diagnosed with recent HCV (n = 226) in England (107), The Netherlands (58), France (12), Germany (25), and Australia (24) between 2000 and 2006 were enrolled into a molecular phylogenetic study. Using real-time polymerase chain reaction (PCR), the NS5B region of the HCV genome (436 base pair) was amplified, sequenced, and compared with unrelated NS5B sequences. RESULTS: NS5B sequences were obtained from 200 (89%) cases. Circulating HCV genotypes were 1a (59%), 4d (23%), 3a (11%), 1b (5%), and 2b/c (3%). Phylogenetic analysis revealed 156 (78%) sequences that formed 11 clusters (bootstrap value > 70%) containing between 4 and 37 individual sequences. Country mixing was associated with larger cluster size (17 vs 4.5 sequences; P = .03). "Molecular clock" analysis indicated that the majority (85%) of transmissions occurred since 1996. CONCLUSIONS: Phylogenetic analysis revealed a large international network of HCV transmission among HIV-positive MSM. The rapid spread of HCV among neighboring countries is supported by the large proportion (74%) of European MSM infected with an HCV strain co-circulating in multiple European countries, the low evolutionary distances among HCV isolates from different countries, and the trend toward increased country mixing with increasing cluster size. Temporally, this epidemic coincides with the introduction of highly active antiretroviral therapy and associated increases in sexual risk behaviors. International collaborative public health efforts are needed to mitigate HCV transmission among this population.

Unité(s) : Laboratoire de Microbiologie

Risk of Extended Viral Resistance in Human Immunodeficiency Virus-1-Infected Mozambican Children After First-Line Treatment Failure

VAZ P, CHAIX ML, JANI I, MACASSA E, BILA D, VUBIL A, ANDERSON S, ROUZIOUX C, BRIAND N, BLANCHE S

2009 - Pediatr Infect Dis J 28(12):e283-87

BACKGROUND:: Resistant virus may be selected by sub-optimal control of HIV-1 replication during antiretroviral treatment. The incidence and profile of resistance in children receiving World Health Organization-recommended treatment remains to be evaluated on a large scale. GOALS:: Assessment of the frequency and profile of resistant virus in HIV-1-infected children, treated for at least 6 months with stavudine/zidovudine + lamivudine + nevirapine and presenting virological failure in a large access program in Maputo, Mozambique. RESULTS:: Cross-sectional evaluation of plasma HIV-1 viral load (VL) in 495 evaluable children among 512 treated for at least 6 months showed that 360 (72.7%) had a VL of <50 copies/mL of HIV-1 RNA. Genotypic resistance tests were performed in the 84 available samples from the 135 treated children with VL >/=50 copies/mL: 92% of the viruses were resistant to lamivudine and/or nevirapine, and 15% were resistant to stavudine. Twenty children (24%) harbored virus with a extended spectrum of cross-resistance defined as resistance to the 3 drugs of the combination received by the child and/or at least 1 resistance to a drug to which the child had never been exposed (abacavir: 5%, tenofovir: 6%, didanosine: 3.5% and the new generation non nucleoside inhibitor, etravirine: 6%). The only factor identified by multivariate analysis as being associated with this extended resistance profile was the duration of treatment (aOR: 6.67 [95% CI: 1.24-35.93], P = 0.015 for treatment >24 months) with a per month increase of 1.09 (1.02-1.16) P = 0.007. CONCLUSIONS:: Residual viral replication in children receiving stavudine/zidovudine + lamivudine + nevirapine treatment is associated with a time-dependent risk of acquiring cross-resistance, including resistance to drugs currently used for second-line treatment and also to the new generation of non nucleoside reverse transcritpase inhibitors.

Unité(s) : Laboratoire de Microbiologie, Immunologie-Hématologie Pédiatriques

Immunological success is predicted by enfuvirtide but not interleukin-2 therapy in immunodepressed patients

VIARD JP, FAGARD C, CHAIX ML, ROUZIOUX C, BOUTELOUP V, BENTATA M, DE VERDIERE NC, PAHLAVAN G, WEISS L, LEVY Y, CHENE G, GROUP AET

2009 - AIDS 23(11):1383-8

OBJECTIVES: To evaluate the efficacy of adding interleukin-2 (IL-2) to an optimized background treatment in HIV-1 patients with advanced failure. DESIGN: Randomized, open-label, multicentre controlled trial. METHODS: Patients with CD4 T-cell count of less than 200 cells/microl, plasma HIV-1 RNA of more than 10 000 copies/ml and a genotypic sensitivity score showing two or less active drugs were randomized to either eight IL-2 cycles with optimized background treatment or optimized background treatment alone. Optimization was made according to genotypic sensitivity score. Enfuvirtide was added in enfuvirtide-naive patients. Evaluation was performed at week 52 on the proportions of patients with CD4 cell count of at least 200 cells/microl (primary outcome), of patients with a CD4 cell count increase of at least 50 cells/microl from week 0, on plasma HIV-1 RNA and HIV-related events. RESULTS: Fifty-six patients were analysed. Median age was 43 years, 61% were at Center for Disease Control and Prevention stage C, 43% had a genotypic sensitivity score of 0, median baseline CD4 cell count and plasma HIV-1 RNA values were 64 cells/microl and 4.9 log10 copies/ml, respectively. Treatment could be optimized in 23 patients. At week 52, in the IL-2 and control groups, the proportion of patients with CD4 cell count of at least 200 cells/microl (14 and 18%) or a CD4 cell count increase of at least 50 cells/microl (25 and 32%) and median plasma HIV-1 RNA were not significantly different. In multivariate analysis, optimization with enfuvirtide and baseline CD4 cell count were statistically associated with CD4 cell count of at least 200 cells/microl at week 52 (P = 0.003 and P = 0.01). Optimization with enfuvirtide was the only factor associated with a CD4 cell count gain of at least 50 cells/microl (P < 0.001). There was no difference in the rate of AIDS events between groups. CONCLUSION: IL-2 failed to increase CD4 cell count in immunocompromised patients with multiple therapeutic failures. Enfuvirtide use was highly associated with success.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses

[Extension of beta-lactamases producing bacteria is a worldwide concern.]

ZAHAR JR, BILLE E, SCHNELL D, LANTERNIER F, MECHAI F, MASSE V, NASSIF X, LORTHOLARY O

2009 - Med Sci (Paris) 25(11):939-944

Since 2000's, community extended-spectrum beta-lactamases (ESBL) producing bacteria have spread worldwide, i.e. mostly Escherichia coli that produce ESBL such as CTX-M enzymes. Previous cephalosporins and fluoroquinolones usage are the two most frequent risk factors identified in patients that harbor ESBL-producing bacteria. In addition surveys have shown an alarming trend of associated resistance to others classes of antimicrobial agents among isolates. The emergence of ESBL-producing isolates limits the therapeutic options considerably. For serious systemic infections caused by ESBL-producing bacteria, carbapenems should be regarded as drugs of choice. Preventing the spread and appropriately managing these infections caused by community acquired ESBL producing bacteria have become mandatory.

Unité(s) : Laboratoire de Microbiologie, Maladies Infectieuses

Predicting the risk of documented ventilator-associated pneumonia for benchmarking: Construction and validation of a score*

ZAHAR JR, NGUILE-MAKAO M, FRANCAIS A, SCHWEBEL C, GARROUSTE-ORGEAS M, GOLDGRAN-TOLEDANO D, AZOULAY E, THUONG M, JAMALI S, COHEN Y, DE LASSENCE A, TIMSIT JF

2009 - Crit Care Med 37(9):2545-2551

OBJECTIVES:: To build and validate a ventilator-associated pneumonia risk score for benchmarking. The rate of ventilator-associated pneumonia varies widely with case-mix, a fact that has limited its use for measuring intensive care unit performance. METHODS:: We studied 1856 patients in the OUTCOMEREA database treated at intensive care unit admission by endotracheal intubation followed by mechanical ventilation for >48 hrs; they were allocated randomly to a training data set (n = 1233) or a validation data set (n = 623). Multivariate logistic regression was used. Calibration of the final model was assessed in both data sets, using the Hosmer-Lemeshow chi-square test and receiver operating characteristic curves. MEASUREMENTS AND MAIN RESULTS:: Independent risk factors for ventilator-associated pneumonia were male gender (odds ratio = 1.97, 95% Confidence Interval = 1.32-2.95); SOFA at intensive care unit admission (<3 (1), 3-4 (2.57, 1.39-4.77), 5-8 (7.37, 4.24-12.81), >8 (5.81 (3.2-10.52)), no use within 48 hrs after intensive care unit admission of parenteral nutrition (2.29, 1.52-3.45), no broad-spectrum antimicrobials (2.11, 1.46-3.06); and mechanical ventilation duration (<5 days (1); 5-7 days (17.55, 4.01-76.85); 7-15 days (53.01, 12.74-220.56); >15 days (225.6, 54.3-936.7). Tests in the training set showed good calibration and good discrimination (area under the curve-receiver operating characteristic curve = 0.881), and both criteria remained good in the validation set (area under the curve-receiver operating characteristic curve = 0.848) and good calibration (Hosmer-Lemeshow chi-square = 9.98, p = .5). Observed ventilator-associated pneumonia rates varied across intensive care units from 9.7 to 26.1 of 1000 mechanical ventilation days but the ratio of observed over theoretical ventilator-associated pneumonia rates was >1 in only two intensive care units. CONCLUSIONS:: The ventilator-associated pneumonia rate may be useful for benchmarking provided the ratio of observed over theoretical rates is used. External validation of our prediction score is needed.

Unité(s) : Laboratoire de Microbiologie

Stimulation of the primary anti-HIV antibody response by IFN-{alpha} in patients with acute HIV-1 infection

ADALID-PERALTA L, GODOT V, COLIN C, KRZYSIEK R, TRAN T, POIGNARD P, VENET A, HOSMALIN A, LEBON P, ROUZIOUX C, CHENE G, EMILIE D, THE INTERPRIM ANRS STUDYGROUP

2008 - J. Leukocyte Biol. 83(4):1060-1067

Type I IFNs are needed for the production of antiviral antibodies in mice; whether they also stimulate primary antibody responses in vivo during human viral infections is unknown. This was assessed in patients acutely infected with HIV-1 and treated with IFN-alpha2b. Patients with acute HIV-1 infection were randomized to receive antiretroviral therapy alone (Group A, n=60) or combined for 14 weeks with pegylated-IFN-alpha2b (Group B, n=30). Emergence of anti-HIV antibodies was monitored during 32 weeks by Western blot (WB) analyses of serum samples. IFN-alpha2b treatment stimulated the production of anti-HIV antibodies. On Week 32, 19 weeks after the last IFN-alpha2b administration, there were 8.5 (6.5-10.0) HIV WB bands (median, interquartile range) in Group B and 7.0 (5.0-10.0) bands in Group A (P=0.054), and band intensities were stronger in Group B (P<0.05 for p18, p24, p34, p40, and p55 HIV antigens). IFN-alpha2b treatment also increased circulating concentrations of the B cell-activating factor of the TNF family (P<0.001) and ex vivo production of IL-12 (P<0.05), reflecting its effect on innate immune cells. Withdrawal of antiretroviral treatment on Week 36 resulted in a lower rebound of HIV replication in Group B than in Group A (P<0.05). Therefore, type I IFNs stimulate the emerging anti-HIV immune response in patients with acute HIV-1 infection, resulting in an improved control of HIV replication. Type I IFNs are thus critical in the development of efficient antiviral immune responses in humans, including the production of antiviral antibodies.

Unité(s) : Laboratoire de Microbiologie, EA3620

HIV-1 DNA for the measurement of the HIV reservoir is predictive of disease progression in seroconverters whatever the mode of result expression is

AVETTAND-FENOEL V, BOUFASSA F, GALIMAND J, MEYER L, ROUZIOUX C

2008 - J. Clin. Virol. 42(4):399-404

BACKGROUND: HIV-1 DNA levels, reported as copies/10(6) peripheral blood mononuclear cells (PBMC), are very predictive of disease progression in seroconverters, independently of CD4(+)T cell count and HIV-RNA. Previously, HIV-DNA levels have sometimes been reported by other means: copies/10(6) CD4(+)T cells, reflecting the proportion of infected cells; or copies/mL whole blood, reflecting the global blood reservoir size. OBJECTIVES: We investigated if the predictive value over the natural course of the disease depends on how the results are reported. STUDY DESIGN: Results reported as HIV-DNA copies/10(6) PBMC were converted to copies/10(6) CD4(+)T cells or to copies/mL whole blood for 422 seroconverters included in the French SEROCO cohort (ANRS). RESULTS: The three methods for reporting HIV-DNA levels yielded different ranges, but these values were highly correlated. The level of HIV-DNA during the seroconversion period was strongly associated with disease progression in all three reporting methods. CONCLUSIONS: This reinforces the value of HIV-DNA quantification in physiopathological and therapeutical studies, particularly in an era of research aimed at diminishing the HIV reservoir. Even if blood represents a small part of this reservoir, HIV-DNA in blood is a simple marker that provides an informative picture of the global reservoir and is strongly predictive of disease progression.

Unité(s) : Laboratoire de Microbiologie

HIV-DNA in rectal cells is well correlated with HIV-DNA in blood in different groups of patients, including long-term non-progressors

AVETTAND-FENOEL V, PRAZUCK T, HOCQUELOUX L, MELARD A, MICHAU C, KERDRAON R, AGOUTE E, ROUZIOUX C

2008 - AIDS 22(14):1880-1882

Most of the body's lymphoid tissue is in gut and constitutes an immense HIV reservoir. We quantified HIV-DNA in rectum and compared it with blood levels for 27 HIV-infected adults from different groups. We observed a large range of rectal and blood HIV-DNA levels. They were positively correlated (r = 0.841, P<0.0001). Long-term nonprogressors and patients in 'remission' after antiretroviral treatment interruption had the lowest blood and mucosal HIV-DNA levels.

Unité(s) : Laboratoire de Microbiologie

Incidence of cancer in children perinatally exposed to nucleoside reverse transcriptase inhibitors

BENHAMMOU V, WARSZAWSKI J, BELLEC S, DOZ F, ANDRE N, LACOUR B, LEVINE M, BAVOUX F, TUBIANA R, MANDELBROT L, CLAVEL J, BLANCHE S

2008 - AIDS 22(16):2165-2177

CONTEXT:: Long-term studies of tolerance to perinatal exposure to antiretroviral nucleoside reverse transcriptase inhibitors are required, in view of the potential genotoxicity of some of these molecules. OBJECTIVE:: To evaluate the incidence of cancers in uninfected children born to HIV-infected mothers. METHOD:: Cancers were detected in a nationwide prospective cohort of children born to HIV-infected mothers by standardized questionnaire during the prospective follow-up period of 2 years; thereafter, they were detected by spontaneous pharmacovigilance declaration and by crosschecking data with the national registries of childhood cancer. Standardized incidence ratio for incidence comparisons with general population. RESULTS:: Ten cases of cancer were detected among the 9127 exposed HIV-uninfected children (median age: 5.4 years, 53 052 person-years of follow-up). The overall incidence did not differ significantly from that expected for the general population: 10 cases observed versus 8.9 and 9.6 expected depending on whether 1990-1999 or 2000-2004 national rates were used as reference [standardized incidence ratio of 1.1 (0.3-1.5) and 1.0 (0.5-1.9)]. Five cases of central nervous system cancer were observed (standardized incidence ratio of 3.1 [1.0-7.2] P = 0.05 and 2.4 [0.8-5.6], P = 0.12). The relative risk of cancer for children exposed to didanosine-lamivudine combination was higher than that for zidovudine monotherapy [hazard ratio: 13.6 (2.5-73.9)]. CONCLUSION:: This study did not evidence an overall increase in cancer risk in nucleoside reverse transcriptase inhibitor exposed children until 5 years of age. Results suggesting associations with specific nucleoside reverse transcriptase inhibitor combinations need further investigations. A longer surveillance, including differential analysis of the different cancer sites and various nucleoside reverse transcriptase inhibitors administered is warranted.

Unité(s) : Laboratoire de Microbiologie, Immuno-Hématologie-Rhumatologie Pédiatriques, EA3620

Candidemia and candiduria in critically ill patients admitted to intensive care units in France: incidence, molecular diversity, management and outcome

BOUGNOUX ME, KAC G, AEGERTER P, D'ENFERT C, FAGON JY

2008 - Intens. Care Med. 34(2):292-299

OBJECTIVE: To determine the concomitant incidence, molecular diversity, management and outcome of nosocomial candidemia and candiduria in intensive care unit (ICU) patients in France. DESIGN: A 1-year prospective observational study in 24 adult ICUs. PATIENTS: Two hundred and sixty-two patients with nosocomial candidemia and/or candiduria. MEASUREMENTS AND RESULTS: Blood and urine samples were collected when signs of sepsis were present. Antifungal susceptibility of Candida strains was determined; in addition, all blood and 72% of urine C. albicans isolates were analyzed by using multi-locus sequence type (MLST). The mean incidences of candidemia and candiduria were 6.7 and 27.4/1000 admissions, respectively. Eight percent of candiduric patients developed candidemia with the same species. The mean interval between ICU admission and candidemia was 19.0[Symbol: see text]+/-[Symbol: see text]2.9 days, and 17.2[Symbol: see text]+/-[Symbol: see text]1.1 days for candiduria. C. albicans and C. glabrata were isolated in 54.2% and 17% of blood and 66.5% and 21.6% of urine Candida-positive cultures, respectively. Fluconazole was the most frequently prescribed agent. In all candidemic patients, the prescribed curative antifungal agent was active in vitro against the responsible identified strain. Crude ICU mortality was 61.8% for candidemic and 31.3% for candiduric patients. Seventy-five percent of the patients were infected with a unique C. albicans strain; cross-transmission between seven patients was suggested in one hospital. CONCLUSIONS: Candidemia is late-onset ICU-acquired infection associated with high mortality. No difference in susceptibility and genetic background were found between blood and urine strains of Candida species.

Unité(s) : Laboratoire de Microbiologie

Mating is rare within as well as between clades of the human pathogen Candida albicans

BOUGNOUX ME, PUJOL C, DIOGO D, BOUCHIER C, SOLL DR, D'ENFERT C

2008 - Fungal. Genet. Biol. 45(3):221-231

Candida albicans is a diploid yeast that can undergo mating and a parasexual cycle, but is apparently unable to undergo meiosis. Characterization of the population structure of C. albicans has shown that reproduction is largely clonal and that mating, if it occurs, is rare or limited to genetically related isolates. Because molecular typing has delineated distinct clades in C. albicans, we have tested whether recombination was common within clades, but rare between clades. Two hundred and three C. albicans isolates have been subjected to multilocus sequence typing (MLST) and the haplotypes at heterozygous MLST genotypes characterized. The C. albicans isolates were distributed among nine clades, of which five corresponded to those previously identified by Ca3 fingerprinting. In each of these clades with more than 10 isolates, polymorphic nucleotide positions located on between 3 and 4 of the six loci were in Hardy-Weinberg disequilibrium. Moreover, each of these polymorphic sites contained excess heterozygotes. This was confirmed by an expanded analysis performed on a recently published MLST dataset for 1044 isolates. On average, 66% of polymorphic positions in the individual clades were in significant excess of heterozygotes over the five clades. These data indicate that mating within clades as well as self-fertilization are both limited and that C. albicans clades do not represent a collection of cryptic species. The study of haplotypes at heterozygous loci performed on our dataset indicates that loss of heterozygosity events due to mitotic recombination is moderately common in natural populations of C. albicans. The maintenance of substantial heterozygosity despite relatively frequent loss of heterozygosity could result from a selective advantage conferred by heterozygosity.

Unité(s) : Laboratoire de Microbiologie

Characteristics of the env Genes of HIV Type 1 Quasispecies in Long-Term Nonprogressors With Broadly Neutralizing Antibodies

BRAIBANT M, AGUT H, ROUZIOUX C, COSTAGLIOLA D, AUTRAN B, BARIN F

2008 - JAIDS 47(3):274-284

Primary isolates of different subtypes of HIV-1 can be neutralized in vitro by the broadly neutralizing antibodies (NAbs) found in the sera of a small number of HIV-1-infected patients. This broad response is most frequent in long-term nonprogressors (LTNPs). We investigated whether the presence of NAbs in the sera of some LTNPs was associated with particular properties of the envelope glycoproteins of the variants found in these patients. Toward that aim, 147 env gene fragments (encoding almost the entire gp120) amplified from the proviral DNA of 5 LTNPs who developed broadly NAbs (NAb+) and of 4 LTNPs who did not develop such broadly NAbs (NAb-) were cloned, sequenced, and compared. We found that the development of broadly NAbs was associated with high viral loads, greater diversity in the gp120 of the viruses infecting these patients, and longer V1 sequences and additional N-gly sites in V1. In addition, a higher proportion of defective clones was found among the env genes of NAb-patients (25% to 93%)-particularly those with lower viral loads and low levels of env diversity-than among those of NAb+ patients (7% to 19%).

Unité(s) : Laboratoire de Microbiologie

Cross-clade-specific cytotoxic T lymphocytes in HIV-1-infected children (vol 250, pg 316, 1998)

BUSEYNE F, CHAIX ML, FLEURY B, MANIGART O, BURGARD M, BLANCHE S, ROUZIOUX C, RIVIRE Y

2008 - Virology 372(2):457

Unité(s) : Département de Pédiatrie, Laboratoire de Microbiologie

Switching to darunavir/ritonavir achieves viral suppression in patients with persistent low replication on first-line lopinavir/ritonavir

CHAIX ML, SAHALI S, PALLIER C, BARRAIL-TRAN A, DELFRAISSY JF, GHOSN J

2008 - AIDS 22(17):2405-2407

Unité(s) : Laboratoire de Microbiologie, EA3620

Coccidioidomycosis: An imported invasive fungal disease in France

CHANDESRIS MO, HOT A, DANNAOUI E, BOUGNOUX ME, VIARD JP, DUPONT B, LORTHOLARY O

2008 - Méd. Mal. Infec. 38(6):336-342

Coccidioidomycosis is an endemic mycosis in the southwest of United States resulting from the inhalation of arthrospores present in desert soil. The authors present a case of uncomplicated pulmonary coccidioidomycosis in a healthy woman, acquired during a recent trip to California. The initial clinical presentation first suggested a diagnosis of community-acquired pneumonia, then of tuberculosis. The diagnosis was finally reached with blood tests and mycological culture of broncho-alveolar lavage fluid. The final identification of Coccidioides immitis was made by molecular analysis. Clinical resolution of the infection was obtained after three months of posaconazole treatment. Coccidioidomycosis is a major cause of pneumonia. Its diagnosis requires specific investigation such as mycological culture, histology, blood tests and molecular biology helps to identify the species. The progression of the disease as well as the associated immunocellular deficit are strictly correlated with the onset of complications and late relapses despite an adequate initial treatment using antifungal molecules and/or surgery.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

Imported disease

CHANDESRIS MO, HOT A, DANNAOUI E, BOUGNOUX ME, VIARD JP, DUPONT B, LORTHOLARY O

2008 - Méd. Mal. Infec. 38(6):291-292

Unité(s) : Infectiologie, Laboratoire de Microbiologie

Maternal 12-Month Response to Antiretroviral Therapy following Prevention of Mother-to-Child Transmission of HIV Type 1, Ivory Coast, 2003-2006

COFFIE PA, EKOUEVI DK, CHAIX ML, TONWE-GOLD B, CLARISSE AB, BECQUET R, VIHO I, N'DRI-YOMAN T, LEROY V, ABRAMS EJ, ROUZIOUX C, DABIS F

2008 - Clin. Infect. Dis. 46(4):611-621

Objective. @nbsp; Our aim was to study the response to antiretroviral treatment among women exposed to single-dose nevirapine (NVP) and/or short-course zidovudine (ZDV; with or without lamivudine [3TC]) for the prevention of mother-to-child transmission of human immunodeficiency virus (HIV) infection. Methods. @nbsp; All HIV type 1-infected women who initiated antiretroviral treatment with stavudine or ZDV, 3TC, and NVP or efavirenz were eligible for the MTCT-Plus program in Abidjan, Ivory Coast. Exposed women had received either single-dose NVP alone or short-course ZDV (with or without 3TC) plus single-dose NVP during previous pregnancy. Genotypic resistance testing was performed at week 4 after delivery. Virologic failure was defined as a plasma HIV RNA level >500 copies/mL 12 months after initiation of antiretroviral treatment. Results. @nbsp; Among 247 women who received antiretroviral treatment, 109 (44%) were unexposed; 81 had received short-course ZDV with 3TC, as well as single-dose NVP; 5 had received short-course ZDV plus 3TC; 50 had received short-course ZDV plus single-dose NVP; and 2 had received single-dose NVP alone. No ZDV mutation was detected in the 115 women whose specimens were available for genotypic testing; 11 (15.1%) of 73 women with 3TC exposure who were tested after delivery had 3TC resistance mutations. Three (4.3%) of 69 women exposed to short-course ZDV and 3TC plus single-dose NVP and 16 (38.1%) of 42 women exposed to short-course ZDV plus single-dose NVP had NVP resistance mutations. Antiretroviral treatment was initiated a median of 21 months after the intervention to prevent mother-to-child HIV transmission (median CD4(+) T lymphocyte count, 188 cells/mm(3)). Month 12 virologic failure was identified in 42 (19.2%) of 219 women for whom data were available, and multivariate analysis revealed that it was associated with poor adherence to treatment (adjusted odds ratio [aOR], 12.7; 95% confidence interval [CI], 3.0-53.9), postpartum 3TC resistance mutations (aOR, 6.9; 95% CI, 1.1-42.9), and a baseline CD4(+) T lymphocyte count <200 cells/mm(3) (aOR, 0.3; 95% CI, 0.2-0.8). NVP resistance was not associated with virological failure (aOR, 1.8; 95% CI, 0.5-6.5). Conclusions. @nbsp; Our study found that poor adherence and 3TC resistance acquired after the intervention to prevent mother-to-child transmission of HIV infection were associated with virologic failure in women who initiated antiretroviral treatment.

Unité(s) : Laboratoire de Microbiologie

One or two enzyme-linked immunosorbent assay tests on the first serum sample for initial diagnosis of HIV-1 infection ?

COSTAGLIOLA D, DAMOND F, PALMER P, ROUZIOUX C, BRUN-VEZINET F

2008 - AIDS 22(15):2042-2044

In France, the first sample for the initial diagnosis of HIV-1 infection must be tested with two antibody assays: one being an enzyme-linked immunosorbent assay. If one is positive, confirmation tests are performed. We evaluated the performance of initial diagnostic strategies based on the use of one versus two enzyme-linked immunosorbent assay tests, either an antigen-antibody test or a simple antibody assay. We found that a single antigen-antibody test was more efficient than a combination of the two tests.

Unité(s) : Laboratoire de Microbiologie, EA3620

Distinct genetic loci control plasma HIV-RNA and cellular HIV-DNA levels in HIV-1 infection: the ANRS Genome Wide Association 01 study

DALMASSO C, CARPENTIER W, MEYER L, ROUZIOUX C, GOUJARD C, CHAIX ML, LAMBOTTE O, AVETTAND-FENOEL V, LE CLERC S, DE SENNEVILLE LD, DEVEAU C, BOUFASSA F, DEBRE P, DELFRAISSY JF, BROET P, THEODOROU I

2008 - PLoS ONE 3(12):e3907

Previous studies of the HIV-1 disease have shown that HLA and Chemokine receptor genetic variants influence disease progression and early viral load. We performed a Genome Wide Association study in a cohort of 605 HIV-1-infected seroconverters for detection of novel genetic factors that influence plasma HIV-RNA and cellular HIV-DNA levels. Most of the SNPs strongly associated with HIV-RNA levels were localised in the 6p21 major histocompatibility complex (MHC) region and were in the vicinity of class I and III genes. Moreover, protective alleles for four disease-associated SNPs in the MHC locus (rs2395029, rs13199524, rs12198173 and rs3093662) were strikingly over-represented among forty-five Long Term HIV controllers. Furthermore, we show that the HIV-DNA levels (reflecting the HIV reservoir) are associated with the same four SNPs, but also with two additional SNPs on chromosome 17 (rs6503919; intergenic region flanked by the DDX40 and YPEL2 genes) and chromosome 8 (rs2575735; within the Syndecan 2 gene). Our data provide evidence that the MHC controls both HIV replication and HIV reservoir. They also indicate that two additional genomic loci may influence the HIV reservoir.

Unité(s) : Laboratoire de Microbiologie

Comparative In Vitro Activities of Caspofungin and Micafungin, Determined Using the Method of the European Committee on Antimicrobial Susceptibility Testing, against Yeast Isolates Obtained in France in 2005-2006

DANNAOUI E, LORTHOLARY O, RAOUX D, BOUGNOUX ME, GALEAZZI G, LAWRENCE C, MOISSENET D, POILANE I, HOINARD D, DROMER F

2008 - Antimicrob. Agents Chemother. 52(2):778-781

The in vitro activities of caspofungin and micafungin against 1,038 yeast isolates have been determined. The caspofungin and micafungin MICs were lower for Candida albicans, Candida glabrata, and Candida tropicalis than for Candida parapsilosis, Candida guilliermondii, and Candida krusei. A clear correlation was seen between the MICs for the two drugs.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

Pneumococcal prophylaxis for children with sickle cell disease in Africa

DE MONTALEMBERT M, BROUSSE V, ZAHAR JR

2008 - Arch. Dis. Child. 93(8):715-716

Unité(s) : Laboratoire de Microbiologie, Pédiatrie Générale

Identification of non fermenting Gram negative bacilli isolated in cystic fibrosis by Matrix assisted laser desorption ionization time-of-flight mass spectrometry

DEGAND N, CARBONNELLE E, DAUPHIN B, BERETTI JL, LE BOURGEOIS M, SERMET-GAUDELUS I, SEGONDS C, BERCHE P, NASSIF X, FERRONI A

2008 - J. Clin. Microbiol. 46(10):3361-3367

The identification of nonfermenting Gram-negative bacilli isolated from cystic fibrosis (CF) patients is usually achieved using phenotypic based techniques and eventually molecular tools. These techniques remain time consuming, expensive, and technically demanding. We used a method based on Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass spectrometry (MALDI-TOF-MS) for the identification of these bacteria. A set of reference strains belonging to 58 species of clinically relevant nonfermenting Gram-negative bacilli was used. To identify peaks discriminating between these various species, the profile of 10 isolated colonies obtained from 10 different passages was analyzed for each referenced strain. Conserved peaks with a relative intensity above 0.1 were retained. The spectra of 559 clinical isolates were then compared with that of each of the 58 reference strains : 400 Pseudomonas aeruginosa, 54 Achromobacter xylosoxydans, 32 Stenotrophomonas maltophilia, 52 Burkholderia cepacia complex (Bcc), 1 Burkholderia gladioli, 14 Ralstonia mannitolilytica, 2 Ralstonia picketti, 1 Bordetella hinzii, 1 Inquilinus limosus, 1 Cupriavidus respiraculi, 1 Burkholderia thailandensis. Using this database, 549 strains were correctly identified. Nine Bcc and 1 R. mannnitolilytica strains were identified as belonging to the appropriate genus but not the correct species. We subsequently engineered Bcc and Ralstonia specific databases using additional reference strains: using these databases, correct identification for these species increased from 83 to 98% and 94 to 100 % of cases, respectively. Altogether, these data demonstrates that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting Gram-negative bacilli.

Unité(s) : Laboratoire de Microbiologie, Pédiatrie Générale, Pneumologie et Asthmologie Pédiatriques

National survey of the prevalence and conditions of selection of HIV-1 reverse transcriptase K70E mutation

DELAUGERRE C, FLANDRE P, MARCELIN AG, DESCAMPS D, TAMALET C, COTTALORDA J, SCHNEIDER V, YERLY S, LEGOFF J, MORAND-JOUBERT L, CHAIX ML, COSTAGLIOLA D, CALVEZ V

2008 - J. Med. Virol. 80(5):762-765

Tenofovir disoproxil fumarate (TDF) has become an important component of HIV combination therapy because of its potency and once-daily dosing. Key mutation associated with resistance to TDF is a K65R in the reverse transcriptase (RT) gene. According to occurrence of K70E mutation after failure to TDF regimen, this mutation was recently reported as a mutation associated with TDF resistance in most resistance genotypic algorithms. The aim of this study was to analyze, retrospectively, the prevalence and conditions of selection of HIV-1 RT K70E mutation from a national clinical survey. Absence of selection of K70E in 850 HIV-1-infected naive patients suggests its role in NRTI drug resistance. Prevalence of K70E RT was low (99/41601, 0.24%) in patients treated between 1999 and 2005. Conversely with K65R mutation, thymidine analog mutations (TAMs) can be concomitantly observed with K70E mutation but its frequency decreased as the number of TAM increases. Concomitant association of K65R and K70E was possible but infrequent (11%). At the time of K70E selection, 60% of patients had received or received TDF-containing regimen and one-third received exclusive NRTI regimen. In conclusion, the K70E mutation could be an alternative pathway of TDF resistance, but as the K65R mutation, other NRTI as ABC, ddI, and 3TC could be also associated with the K70E selection. J. Med. Virol. 80:762-765, 2008. (c) 2008 Wiley-Liss, Inc.

Unité(s) : Laboratoire de Microbiologie

HIV-1-infected patients from the French National Observatory experiencing virological failure while receiving enfuvirtide

DESCAMPS D, ASSOUMOU L, MASQUELIER B, MARCELIN AG, SAIDI S, TAMALET C, COTTALORDA J, PLANTIER JC, MONTES B, IZOPET J, PEYTAVIN G, YERLY S, SCHNEIDER V, DELAUGERRE C, FERRE V, RUFFAULT A, PALLIER C, MORAND-JOUBERT L, CHAIX ML, CALVEZ V, BRUN-VEZINET F, COSTAGLIOLA D

2008 - J. Antimicrob. Chemother. 62(3):451-455

OBJECTIVES: We studied gp41 mutations associated with failing enfuvirtide salvage therapy. METHODS: This multicentre study involved patients with HIV-1 plasma viral load (pVL) > 5000 copies/mL after at least 3 months of uninterrupted enfuvirtide therapy and with plasma samples available at inclusion (T0), at initial enfuvirtide failure (T1) and at last follow-up visit during continued failing enfuvirtide therapy (T2). The HR-1 and HR-2 domains of the gp41 gene were sequenced at T0, T1 and T2. RESULTS: Ninety-nine patients were enrolled. At baseline, the median pVL and CD4 cell count were 5.1 log copies/mL and 72 cells/mm(3), respectively. Based on the ANRS Resistance Group algorithm, the proportion of patients harbouring viruses with enfuvirtide resistance mutations increased significantly between T0 and T1. In the HR-1 domain, the V38A/M, Q40H, N42T, N43D and L45M mutations wereselected (P < 0.02). In the HR-2 domain, no mutations were significantly selected during the follow-up. None of the mutations was associated with a CD4 cell count increment. CONCLUSIONS: Mutations selected during failing enfuvirtide salvage therapy are mainly located in the HR-1 domain of the gp41 gene, between codons 38 and 45. No mutations were associated with an increase in the CD4 cell count.

Unité(s) : Laboratoire de Microbiologie

Evaluation of a new commercial real-time PCR quantification assay for prenatal diagnosis of cytomegalovirus congenital infection

DUCROUX A, CHERID S, BENACHI A, VILLE Y, LERUEZ-VILLE M

2008 - J. Clin. Microbiol. 46(6):2078-2080

A new commercial real time HCMV PCR kit was evaluated after automated DNA extraction of 153 amniotic fluids in parallel with an in house real time PCR assay. The commercial kit displayed 100% sensitivity/specificity compared to the "in house" assay and was suitable for prenatal diagnosis of HCMV congenital infection.

Unité(s) : Laboratoire de Microbiologie, Obstétrique

Two cases of Bacillus infection and immunodepression

FARHAT H, CHACHATY E, ANTOUN S, NITENBERG G, ZAHAR JR

2008 - Méd. Mal. Infec. 38(11):612-614

OBJECTIVE: Members of the genus Bacillus are Gram-positive bacilli, ubiquitous in the environment. When isolated in clinical practice, it is frequently considered as due to environmental contamination. Bacillus cereus is the most frequent species isolated in clinical practice, nevertheless other Bacillus spp. are sometimes isolated. Bacillus bacteremia is uncommon, the affected patients are severely ill and frequently immunocompromised with hematological malignancies. STUDY DESIGN: Two cases of bloodstream infection due to Bacillus species rarely described before are described, one due to Bacillus macerans and the other to Bacillus pumilus. Both patients presented with severe bacteremia and were immunodepressed after recent chemotherapy. They died a few days after admission to our ICU. CONCLUSION: The initial report of Bacillus spp. isolated in blood culture in oncohematological patients indicates a potentially severe infection.

Unité(s) : Laboratoire de Microbiologie

Bacterial contamination in the environment of hospitalised children with cystic fibrosis

FERRONI A, WERKHAUSER-BERTRAND A, LE BOURGEOIS M, BEAUVAIS R, VRIELYNCK S, DURAND C, LENOIR G, BERCHE P, SERMET-GAUDELUS I

2008 - J. Cyst. Fibrosis 7(6):477-482

Pathogenic bacterial colonisation in Cystic Fibrosis patients is associated with a poor prognosis; thus, protective measures need to be taken to prevent their transmission. We studied the extent of contamination in the environment of hospitalised children with cystic fibrosis (CF) associated with specific activities. We assessed the levels of bacterial contamination in 432 air and surface samples collected from various locations in our CF centre over a three-month period: the bedrooms, corridor, communal showers, school, leisure centre and the respiratory functional explorations (RFE) unit. Staphylococcus aureus and Pseudomonas aeruginosa strains found in bedrooms and the RFE were compared with those found in patient expectorations using pulsed field gel electrophoresis. In all sampling locations, there were high levels of airborne contamination just after the presence of patients or nursing staff. In the bedrooms, the amount of S. aureus or P. aeruginosa in the air, at wake-up and after physiotherapy, were significantly higher than that after the bedroom had been cleaned. For P. aeruginosa, 33% of isolates were multiresistant to antibiotics; 50% of the colonised patients had the same P. aeruginosa strain in their sputum as in air taken from their bedroom. P. aeruginosa was detected in 23% of samples taken from the surfaces in the showers after patient washing. Very low levels of pathogenic bacteria were found in samples from the other locations. Overall, activities with the highest risk of contamination in the CF ward are physiotherapy and washing in the communal shower room. We therefore recommend to open windows after physiotherapy and to implement a strong decontamination after showers.

Unité(s) : Laboratoire de Microbiologie, Pédiatrie Générale, Pneumologie et Asthmologie Pédiatriques

New and old complex recombinant HIV-1 strains among patients with primary infection in 1996-2006 in France: the French ANRS CO06 primo cohort study

FRANGE P, GALIMAND J, VIDAL N, GOUJARD C, DEVEAU C, SOUALA F, PEETERS M, MEYER L, ROUZIOUX C, CHAIX ML

2008 - Retrovirology 5(.):69

BACKGROUND: Prevalence of HIV-1 non-B subtypes has increased overtime in patients diagnosed at the time of primary infection (PHI) in France. Our objective was to characterize in detail non-B strains which could not be genetically classified into the known subtypes/Circulating Recombinant Forms (CRFs). METHODS: Among 744 patients enrolled in the ANRS PRIMO Cohort since 1996, 176 (23.7%) were infected with HIV-1 non-B strains. The subtype/CRF could not be identified in RT for 15 (2%). The V3-V5 env region was sequenced and 3 strains (04FR-KZS, 06FR-CRN, 04FR-AUK) were full-length sequenced. Phylogenetic and bootscan analyses were used to characterize the mosaic structures. RESULTS: Among V3-V5 sequences, 6 were divergent A, 2 distantly related to E or D, 2 C, 1 B and 2 remained unclassified. 04FR-KZS, isolated in a Congolese woman infected in France, clustered with 2 previously described viruses from the Democratic Republic of Congo. They represent CRF27_cpx involving A/E/G/H/J/K/U subtypes. 06FR-CRN, isolated in a homosexual Caucasian patient, was a B/C/U recombinant involving a Brazilian C strain. 04FR-AUK, isolated in a Congolese patient infected in France, was a A/K/CRF09/U recombinant clustering from gag to vif with HIV-1 MAL. Others PHI were further observed in 2006-2007 with 1 KZS and 5 CRN-like viruses, suggesting their spread in France. CONCLUSION: This study illustrates the increasing HIV-1 diversity in France associating new (06FR-CRN) and old (CRF27_cpx and "MAL-like" 04FR-AUK) strains, which are rare in their region of origin but may have a possible founder effect in France. Our results strengthen the French guidelines recommending viro-epidemiological surveillance of HIV-1 diversity.

Unité(s) : Laboratoire de Microbiologie, EA3620

Fibrodysplasia ossificans progressiva

GEHANNO P, BERCHE P, HERCOT O, D'ARRAS L, CABRILLAC-RIVES S, DEROBERT E, CHONE C, KAPLAN FS, LE MERRER M, GLASER DL, PIGNOLO RJ, GOLDSBY RE, KITTERMAN JA, GROPPE J, SHORE EM

2008 - Méd. Mal. Infec. 38(2P2):8

Fibrodysplasia ossificans progressiva (FOP), a rare and disabling genetic condition of congenital skeletal malformations and progressive heterotopic ossification (HO), is the most catastrophic disorder of HO in humans. Episodic disease flare-ups are precipitated by soft tissue injury, and immobility is cumulative. Recently, a recurrent mutation in activin receptor IA/activin-like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor, was reported in all sporadic and familial cases of classic FOP, making this one of the most highly specific disease-causing mutations in the human genome. The discovery of the FOP gene establishes a critical milestone in understanding FOP, reveals a highly conserved target for drug development in the transforming growth factor (TGF)-beta/BMP signalling pathway, and compels therapeutic approaches for the development of small molecule signal transduction inhibitors for ACVR1/ALK2. Present management involves early diagnosis, assiduous avoidance of iatrogenic harm, and symptomatic amelioration of painful flare-ups. Effective therapies for FOP, and possibly for other common conditions of HO, may potentially be based on future interventions that block ACVR1/ALK2 signalling.

Unité(s) : Laboratoire de Microbiologie, Génétique Médicale Pédiatrique

Absence of HIV-1 shedding in male genital tract after 1 year of first-line lopinavir/ritonavir alone or in combination with zidovudine/lamivudine

GHOSN J, CHAIX ML, PEYTAVIN G, BRESSON JL, GALIMAND J, GIRARD PM, RAFFI F, COHEN-CODAR I, DELFRAISSY JF, ROUZIOUX C

2008 - J. Antimicrob. Chemother. 61(6):1344-1347

Background New strategies such as boosted-protease inhibitor (PI) monotherapy are being investigated. However, a concern remains regarding the efficacy of this strategy in viral sanctuaries such as the male genital tract. More than 80% of untreated HIV-infected men have detectable HIV-RNA in semen and such a strategy could favour local selection of resistant variants, given the poor penetration of most PIs in semen. Objectives To evaluate the impact of a first-line lopinavir/ritonavir alone or standard triple combination on HIV-1 shedding in the genital tract. Methods HIV-1-infected men enrolled in the Monark randomized trial were eligible for the present study after 48 weeks of a first-line lopinavir/ritonavir alone or in combination with zidovudine and lamivudine. Single-paired samples of blood and semen were collected at week 48. Blood plasma HIV-RNA and seminal plasma HIV-RNA were measured at week 48. Lopinavir and ritonavir concentrations were measured in blood and in semen at week 48 by high-performance liquid chromatography. Results Ten patients were included: five of them received lopinavir/ritonavir monotherapy and five received a triple combination. At week 48, all patients had blood plasma HIV-RNA <1.7 log(10) copies/mL. Median lopinavir and ritonavir concentrations were within the expected therapeutic target range in blood plasma (4896 and 130.5 ng/mL, respectively), whereas both lopinavir and ritonavir were undetectable in all seminal plasma samples (<30 ng/mL). All 10 patients had undetectable seminal plasma HIV-RNA at week 48 (<2.3 log(10) copies/mL). Conclusions No local viral production was evident in semen, despite the local absence of therapeutic antiretroviral drug concentrations in the five patients receiving lopinavir/ritonavir alone.

Unité(s) : Laboratoire de Microbiologie, CIC 9303, EA3620

Sexually transmitted hepatitis C virus superinfection in HIV/hepatitis C virus co-infected men who have sex with men

GHOSN J, THIBAULT V, DELAUGERRE C, FONTAINE H, LORTHOLARY O, ROUZIOUX C, POL S, CHAIX ML

2008 - AIDS 22(5):658-661

We report two cases of sexually transmitted hepatitis C virus (HCV) superinfection in HIV/HCV-co-infected patients with high-risk sexual behaviour. The two patients had chronic HCV and a history of sexually transmitted infections. HCV superinfection was confirmed by phylogenetic analysis. No risk factors for HCV were found except unprotected anal sex with multiple casual male partners. HCV serology and serum HCV RNA should be examined periodically in HIV-infected men who have sex with men engaging in high-risk sexual behaviours.

Unité(s) : Infectiologie, Laboratoire de Microbiologie, Hépatologie Adulte, EA3620

Osteoarticular infections: therapeutic proposals of the Paediatric Infectious Diseases Group of the French Society of Paediatrics (GPIP

GRIMPREL E, LORROT M, HAAS H, PINQUIER D, PAREZ N, FERRONI A, COHEN R

2008 - Archives Pédiatrie 15(Suppl.2):S74-S80

The empiric choice of initial antibiotherapy in osteoarticular infections in infants and children must take into consideration the actual epidemiology of principal pathogens, their respective antibiotic sensitivity profile, their pharmacokinetic and pharmacodynamic properties and the results of efficacy clinical studies. After a review of recent data concerning these four major points, the Paediatric Infectious Diseases Group of the French Society of Paediatrics (GPIP) has proposed guidelines for initial recommended schemes of antimicrobial therapy in acute and non complicated osteoarticular infections in infants and children.

Unité(s) : Laboratoire de Microbiologie

Osteoarticular infections: clinical studies

GRIMPREL E, LORROT M, HAAS H, PINQUIER D, PAREZ N, FERRONI A, COHEN R

2008 - Archives Pédiatrie 15(Suppl.2):S68-S73

Management of paediatric skeletal infections remains delicate in France due to the absence of general agreement or official recommendations from the French National Societies. However, practices have evolved since 40 years towards simplified and mostly ambulatory treatment modalities. The Paediatric Infectious Diseases Group of the French Society of Paediatrics (GPIP) has elaborated guidelines funded on the joint analysis of bacterial epidemiology, comparison of common antimicrobial spectra, pharmacokinetic and pharmacodynamic parameters and clinical studies. The aim of this article is to review the evolution of therapeutic concepts of treatment of bone and joint infections in paediatrics for 40 years with the aid of main published clinical studies.

Unité(s) : Laboratoire de Microbiologie

Assisted procreation technology and people with HIV

GUIBERT J, LERUEZ-VILLE M, DULIOUST E, LAUNAY O, SOGNI P, CHARLEMAINE E, ROUZIOUX C, JOUANNET P

2008 - Presse Médicale 37(6P2):998-1006

In France, a ministerial decree dated 10 May 2001 authorizes the use of assisted reproduction technologies (ART) for people infected with the human immunodeficiency virus (HIV), either to reduce the risk of transmission between partners or to treat the couple's infertility. The HIV patient must have a CD4 T lymphocyte count>200/mm(3) and a stable viral load (no increase exceeding 0.5 log(10) copies/mm(3)) between 2 samples during the 6 months preceding ART. Co-infections with hepatitis B or C must be assessed by a specialist. When the man is infected, only ART allows conception while simultaneously ensuring safe sexual relations between the couple. ART is performed with prepared spermatozoa, validated negative for HIV RNA. The particular ART method depends on the results of the couple's fertility assessment and the quantity of virus in the seminal fluid. Antiretroviral treatment is not required for ART but may be necessary if the seminal viral load is elevated. When the woman is infected, the couple must be informed about the risks of HIV transmission to the child and of toxicity to mother and fetus from the antiretroviral treatments. These risks must guide optimization of the antiretroviral treatment, which is not routine during ART but is systematic during the last trimester. Management of pregnancy planning should propose artificial insemination and rapid recourse to ART because ovarian function appears to deteriorate quickly in women with HIV. Several thousand couples in Europe have used ART without any cases of contamination reported so far. Approximately half of these couples can hope to have a child, but approximately one third decide against ART after consultation.

Unité(s) : Laboratoire de Microbiologie

Multilocus sequence typing of Candida albicans isolates from animals

JACOBSEN MD, BOUGNOUX ME, D'ENFERT C, ODDS FC

2008 - Res. Microbiol. 159(6):436-440

Multilocus sequencing strain types of a panel of 43 Candida albicans isolates from animals, including mammals and avian species, were compared with strain types for human isolates. The clade distribution of the animal isolates was significantly different from that of the human isolates, in both a comparison involving a total of 1580 isolates from multiple geographical sources and a comparison restricted to 675 human isolates from the same geographical regions as the animal isolates. A nearest-neighbour analysis involving the 43 animal isolates and 67 human isolates, randomly selected to give a proportionate distribution of geographical sources, showed a strong statistical trend towards genetic selection of different C. albicans strain types adapted to non-human animal hosts, but without complete genetic separation.

Unité(s) : Laboratoire de Microbiologie

Prevention of mother-to-child HIV transmission: similar access for sub-Sahara African immigrants and for French women ?

JASSERON C, MANDELBROT L, TUBIANA R, TEGLAS JP, FAYE A, DOLLFUS C, LE CHENADEC J, ROUZIOUX C, BLANCHE S, WARSZAWSKI J

2008 - AIDS 22(12):1503-1511

OBJECTIVE:: To investigate whether mother-to-child transmission (MTCT) management and rate differed between African immigrants and French-born women delivering in France. METHODS:: MTCT strategies were studied among human immunodeficiency virus type 1-infected women delivering between 1984 and 2007 in the multicenter French Perinatal Cohort, according to geographical origin. RESULTS:: Among 9245 pregnancies (in 7090 women), the proportion of African mothers increased from 12% in 1984-1986 to 64% in 2003-2004. African women had later access to care than French women, even in recent years (1997-2004). They more often discovered their HIV infection during pregnancy (40.6 vs. 11.5%, P < 0.001), started prenatal care in the third trimester (14.1 vs. 9.8%, P < 0.001) and started antiretroviral therapy after 32 weeks gestation (7.6 vs. 4.1%, P < 0.001). The association with late treatment initiation disappeared when adjusted for late HIV diagnosis and prenatal care (adjusted odds ratio 1.0, 95% confidence interval 0.7-1.4). African and French women did not differ in terms of access to highly active antiretroviral therapy, nor for substandard management such as vaginal delivery with uncontrolled viral load, lack of intrapartum and postpartum treatment or breastfeeding. The MTCT rate was higher for African than for French women receiving antiretroviral therapy (1.8 vs. 0.8%, P = 0.02), but the difference was no longer significant after adjustment for main transmission risk factors (adjusted odds ratio = 1.7, 95% confidence interval 0.8-3.7, P = 0.17). MTCT did not differ among 2110 term deliveries with maternal viral load less than 400 copies/ml, (0.8 vs. 0.6%, P = 0.5). CONCLUSION:: African immigrants more often had late HIV screening in pregnancy than French-born women, but had similar access to MTCT prevention, once the infection was diagnosed.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie, EA3620

Herpes-virus infection in patients with langerhans cell histiocytosis: a case-controlled sero-epidemiological study, and in situ analysis

JEZIORSKI E, SENECHAL B, MOLINA TJ, DEVEZ F, LERUEZ-VILLE M, MORAND P, GLORION C, MANSUY L, GAUDELUS J, DEBRE M, JAUBERT F, SEIGNEURIN JM, THOMAS C, JOAB I, DONADIEU J, GEISSMANN F

2008 - PLoS ONE 3(9):e3262

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare disease that affects mainly young children, and which features granulomas containing Langerhans-type dendritic cells. The role of several human herpesviruses (HHV) in the pathogenesis of LCH was suggested by numerous reports but remains debated. Epstein-barr virus (EBV, HHV-4), & Cytomegalovirus (CMV, HHV-5) can infect Langerhans cells, and EBV, CMV and HHV-6 have been proposed to be associated with LCH based on the detection of these viruses in clinical samples. METHODOLOGY: We have investigated the prevalence of EBV, CMV and HHV-6 infection, the characters of antibody response and the plasma viral load in a cohort of 83 patients and 236 age-matched controls, and the presence and cellular localization of the viruses in LCH tissue samples from 19 patients. PRINCIPAL FINDINGS: The results show that prevalence, serological titers, and viral load for EBV, CMV and HHV-6 did not differ between patients and controls. EBV was found by PCR in tumoral sample from 3/19 patients, however, EBV small RNAs EBERs -when positive-, were detected by in situ double staining in bystander B CD20+ CD79a+ lymphocytes and not in CD1a+ LC. HHV-6 genome was detected in the biopsies of 5/19 patients with low copy number and viral Ag could not be detected in biopsies. CMV was not detected by PCR in this series. CONCLUSIONS/SIGNIFICANCE: Therefore, our findings do not support the hypothesis of a role of EBV, CMV, or HHV-6 in the pathogenesis of LCH, and indicate that the frequent detection of Epstein-barr virus (EBV) in Langerhans cell histiocytosis is accounted for by the infection of bystander B lymphocytes in LCH granuloma. The latter observation can be attributed to the immunosuppressive micro environment found in LCH granuloma.

Unité(s) : U838, Anatomie Pathologique, Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie, Traumatologie et Orthopédie Pédiatriques

Les Menaces Biologiques

KORN H, BERCHE P, BINDER P

2008 - Ouvrage 144

Le comité Science et sécurité de l'Académie des sciences s'est régulièrement réuni depuis septembre 2005, a interrogé de nombreux chercheurs, spécialistes de la santé et des sécurités civile et militaire, pour rédiger ce rapport dans le but d'informer non seulement les autorités gouvernementales, mais aussi l'ensemble du corps social sur les usages criminels auxquels peuvent conduire les progrès de la biologie s'ils sont dévoyés en moyens de destruction. Cet ouvrage d'information se veut aussi un ouvrage de recommandations à explorer dans le cadre d'une mise en oeuvre d'une politique gouvernementale de sécurité.

Unité(s) : Laboratoire de Microbiologie

Successful lumbarperitoneal derivation during refractory intracranial pressure due to cryptococcal meningitis, in an HIV-negative patient

LANTERNIER F, LECUIT M, LORTHOLARY O, ROUJEAU T, RENIER D, BOUGNOUX ME

2008 - Méd. Mal. Infec. 38(5):285-286

Unité(s) : Infectiologie, Laboratoire de Microbiologie, Neurochirurgie Pédiatrique

Fusobacterium necrophorum Middle Ear Infections in Children and Related Complications: Report of 25 Cases and Literature Review

LE MONNIER A, JAMET A, CARBONNELLE E, BARTHOD G, MOUMILE K, LESAGE F, ZAHAR JR, MANNACH Y, BERCHE P, COULOIGNER V

2008 - Pediat. Inf. Dis. J. 27(7):613-617

BACKGROUND:: Fusobacterium necrophorum is associated with Lemierre syndrome (pharyngitis with septic thrombosis of the internal jugular veins) but it can also be involved in other head and neck infections, including sinusitis, parotitis, dental infections, and otitis media. METHODS:: This retrospective study analyzes a series of 25 pediatric cases of acute otitis media caused by F. necrophorum and treated in our institution between 1995 and 2006. RESULTS:: We observed 3 clinical presentations: (1) uncomplicated otitis media (44%; n = 11); (2) acute mastoiditis (40%; n = 10); and (3) otogenic variant of Lemierre syndrome (16%; n = 4) associating acute mastoiditis, suppurative thrombophlebitis of the lateral and/or cavernous sinuses, meningitis syndrome, and sometimes distant septic metastasis or extensive osteolysis of the temporal bone. Sixty percent of these cases were diagnosed during the last 4 years of the study. Children less than 1 year of age were at increased risk for Lemierre syndrome. Broad range 16S rDNA polymerase chain reaction and sequencing were used to confirm the identification of F. necrophroum and to detect secondary sites of infection. All patients had favorable clinical outcome, but complicated cases (mastoiditis and otogenic variant of Lemierre syndrome) required prolonged hospital stays and duration of treatment. CONCLUSIONS:: Based on bacteriologic investigation, we recommend systematic culture for anaerobes and that antibiotic treatment of F. necrophorum middle ear infections and subsequent complications includes coverage for anaerobic bacteria.

Unité(s) : Laboratoire de Microbiologie, ORL & Chirurgie Cervico-Faciale, Réanimation Pédiatrique & Néonatologie

Changes in enoxaparin pharmacokinetics during pregnancy and implications for antithrombotic therapeutic strategy

LEBAUDY C, HULOT JS, AMOURA Z, COSTEDOAT-CHALUMEAU N, SERREAU R, ANKRI A, CONARD J, CORNET A, DOMMERGUES M, PIETTE JC, LECHAT P

2008 - Clin. Pharmacol. Ther. 84(3):370-377

Enoxaparin is frequently prescribed for pregnant women who are at high risk for thromboembolic complications. We conducted a population pharmacokinetics study with 75 pregnant women and 38 nonpregnant women as controls to evaluate enoxaparin pharmacokinetics during pregnancy and the postpartum period. Clearance of the drug was higher in the pregnant women throughout pregnancy when compared with nonpregnant women (0.78 +/- 0.03 l/h vs. 0.52 +/- 0.03 l/h, respectively P < 0.001) with the stage of the pregnancy having no influence. The volume of distribution was influenced by stage of the pregnancy, characterized by a two-step increase, with an initial rise paralleling the woman's increase in body weight during the first two trimesters, followed by an additional increase of 41% during the last 2 months of pregnancy, independent of changes in weight. Using enoxaparin pharmacokinetic parameters to simulate anti-Xa time profiles, we observed that the maintenance of the same doses throughout pregnancy resulted in a progressive reduction in mean and peak anti-Xa activities. We recommend the administration of doses normalized for body weight changes so as to counteract enoxaparin pharmacokinetic changes that accompany various stages of pregnancy.

Unité(s) : Laboratoire de Microbiologie, EA3620

Simultaneous determination of three carbapenem antibiotics in plasma by HPLC with ultraviolet detection

LEGRAND T, CHHUN S, REY E, BLANCHET B, ZAHAR JR, LANTERNIER F, PONS G, JULLIEN V

2008 - J. Chromatogr. B (Anal. Technol. Biomed. Life Sci.) 875(2):551-556

A simple, precise and accurate high-performance liquid chromatography (HPLC) method using ultraviolet (UV) detection has been developed for simultaneous determination of carbapenem antibiotics: imipenem, meropenem and ertapenem in human plasma. Samples were spiked with ceftazidime as internal standard and proteins were precipitated by acetonitrile. Separation was achieved on a C8 column with a mobile phase composed of phosphate buffer 0.1M (pH 6.8) and methanol in gradient elution mode. Detection was performed at 298nm. Calibration curves were linear from 0.5 to 80mg/L for each compound, with correlation coefficients over 0.997. Intra- and inter-day validation studies showed accuracy between -4.5 and 8.1% and precision below 10.4%. Mean recoveries were 82.2, 90.8 and 87.7% for imipenem, meropenem and ertapenem, respectively. This method provides a useful tool for the therapeutic drug monitoring of carbapenems.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

18-Month Effectiveness of Short-Course Antiretroviral Regimens Combined with Alternatives to Breastfeeding to Prevent HIV Mother-to-Child Transmission

LEROY V, EKOUEVI DK, BECQUET R, VIHO I, DEQUAE-MERCHADOU L, TONWE-GOLD B, ROUET F, SAKAROVITCH C, HORO A, TIMITE-KONAN M, ROUZIOUX C, DABIS F

2008 - PLoS ONE 3(2):e1645

OBJECTIVE: We assessed the 18-month effectiveness of short-course (sc) antiretroviral peripartum regimens combined with alternatives to prolonged breastfeeding to prevent mother-to-child transmission (MTCT) of HIV-1 in Abidjan, Cote d'Ivoire. METHODOLOGY: HIV-1 infected pregnant women received from >/=32-36 weeks of gestation scZidovudine (ZDV)+/-Lamivudine (3TC)+single-dose Nevirapine (sdNVP) at delivery within the ANRS 1201/1202 DITRAME-Plus cohort (2001-2003). Neonates received a sdNVP+7-day ZDV prophylaxis. Two infant-feeding interventions were systematically offered free of charge: formula-feeding or exclusive shortened breastfeeding with early cessation from four months. The reference group was the ANRS 049a DITRAME cohort (1994-2000) exposed to scZDV from 36 weeks, then to prolonged breastfeeding. Pediatric HIV infection was defined by a positive plasma HIV-1 RNA at any age, or if aged >/=18 months, a positive HIV-1 serology. Turnbull estimates of cumulative transmission risks (CTR) and effectiveness (HIV-free survival) were compared by exposure group using a Cox model. FINDINGS: Among 926 live-born children enrolled, 107 (11.6%) were HIV-infected at 18 months. CTRs were 22.3% (95% confidence interval[CI]:16-30%) in the 238 ZDV long-term breastfed reference group, 15.9% (CI:10-27%) in the 169 ZDV+sdNVP shortened breastfed group; 9.4% (CI:6-14%) in the 195 ZDV+sdNVP formula-fed group; 6.8% (CI:4-11%) in the 198 ZDV+3TC+sdNVP shortened breastfed group, and 5.6% (CI:2-10%) in the 126 ZDV+3TC+sdNVP formula-fed group. Each combination had a significantly higher effectiveness than the ZDV long-term breastfed group except for ZDV+sdNVP shortened breastfed children, ranging from 51% (CI:20-70%) for ZDV+sdNVP formula fed children to 63% (CI:40-80%) for ZDV+3TC+NVPsd shortened breastfed children, after adjustment for maternal eligibility for antiretroviral therapy (ART), home delivery and low birth-weight. Substantial MTCT risk reductions are reachable in Africa, even in short-term breastfed children. The two sc antiretroviral combinations associated to any of the two infant feeding interventions, formula-feeding and shortened breastfeeding, reduce significantly MTCT with long-term benefit until age 18 months and without increasing mortality.

Unité(s) : Laboratoire de Microbiologie

Exon 4 of the human cytomegalovirus (CMV) major immediate-early gene as a target for CMV real-time PCR

LERUEZ-VILLE M, DUCROUX A, ROUZIOUX C

2008 - J. Clin. Microbiol. 46(4):1571-1572

Unité(s) : Laboratoire de Microbiologie

Fournier's gangrene due to Candida glabrata

LOULERGUE P, MAHE V, BOUGNOUX ME, POIREE S, HOT A, LORTHOLARY O

2008 - Med. Mycol. 46(2):171-173

Fournier's gangrene is a rare and serious event. Usual pathogens are bacteria of the skin and the digestive tract. Candida species are exceptionally involved, mostly Candida albicans. We report a patient with non-C. albicans Candida sp. Fournier's gangrene who survived with an appropriate antifungal therapy. Yeasts should be considered as emerging pathogens in pelvic infections due to the increase in long-term immunocompromised patients.

Unité(s) : Anesthésie Réanimation, Infectiologie, Laboratoire de Microbiologie, Radiologie Adulte

Tipranavir-ritonavir genotypic resistance score in protease inhibitor-experienced patients

MARCELIN AG, MASQUELIER B, DESCAMPS D, IZOPET J, CHARPENTIER C, ALLOUI C, BOUVIER-ALIAS M, SIGNORI-SCHMUCK A, MONTES B, CHAIX ML, AMIEL C, SANTOS GD, RUFFAULT A, BARIN F, PEYTAVIN G, LAVIGNON M, FLANDRE P, CALVEZ V

2008 - Antimicrob. Agents Chemother. 52(9):3237-3243

To identify mutations associated with the virological response (VR) to a tipranavir-ritonavir (TPV/r)-based regimen, 143 patients previously treated with protease inhibitor (PI) were studied. VR was defined by a decrease of at least 1 log(10) in, or undetectable, human immunodeficiency virus (HIV) RNA at month 3. The effect of each mutation in the protease, considering all variants at a residue as a single variable, on the VR to TPV/r was investigated. Mutations at six residues were associated with a lower VR (E35D/G/K/N, M36I/L/V, Q58E, Q61D/E/G/H/N/R, H69I/K/N/Q/R/Y, and L89I/M/R/T/V), and one mutation was associated with a higher VR (F53L/W/Y). The genotypic score M36I/L/V-53L/W/Y + Q58E + H69I/K/N/Q/R/Y + L89I/M/R/T/V was selected as providing a strong association with VR. For the seven patients with a genotypic score of -1 (viruses with only mutation at codon 53), the percentage of responders was 100% and the percentages were 79%, 56%, 33%, 21%, and 0% for those with scores of 0, 1, 2, 3, and 4, respectively. The percentage of patients showing a response to TPV/r was lower for patients infected with non-clade B viruses (n = 16, all non-B subtypes considered together) than for those infected with clade B viruses (n = 127) (25% and 59%, respectively; P = 0.015). Most mutations associated with VR to TPV/r had not previously been associated with PI resistance. This is consistent with phenotypic analysis showing that TPV has a unique resistance profile. Mutations at five positions (35, 36, 61, 69, and 89) were observed significantly more frequently in patients infected with a non-B subtype than in those infected with the B subtype, probably explaining the lower VR observed in these patients.

Unité(s) : Laboratoire de Microbiologie

HIV-1 DNA in Peripheral Blood Mononuclear Cells is Strongly Associated With HIV-1 Disease Progression in Recently Infected West African Adults

MINGA AK, ANGLARET X, TONI TD, CHAIX ML, DOHOUN L, ABO Y, COULIBALY A, DUVIGNAC J, GABILLARD D, ROUET F, ROUZIOUX C

2008 - JAIDS 48(3):350-354

OBJECTIVE:: To analyze the association between the HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) and disease progression in recently infected West African adults. METHODS:: HIV-1 DNA levels were measured in the PBMCs of 200 adults in the French National Agency for Research on AIDS and viral Hepatitis (ANRS) 1220 cohort who had recently been infected with HIV-1. The association between baseline HIV-1 DNA levels and disease progression was analyzed using multivariate Cox regression. Disease progression was defined as the occurrence of any of the following outcomes: death, first World Health Organization stage 3-4 event, or CD4 count <200/mm. RESULTS:: About 200 participants were followed for a median of 30 months. At baseline, the median time from HIV-1 seroconversion was 9 months, median CD4 T-cell count was 471/mm, median HIV-1 DNA level was 3.0 log10 copies/10 PBMCs, and median plasma HIV-1 RNA level was 4.6 log10 copies/mL. The 5-year probability of remaining free of any outcome was 0.74 [95% confidence interval (CI): 0.61 to 0.83] and 0.36 (95% CI: 0.23 to 0.49) in patients with baseline HIV-1 DNA 3.0 log10 copies/10 PBMCs, respectively (P < 0.001). The adjusted hazard ratio of disease progression was 2.17 in patients with HIV-1 DNA >3.0 log10 copies/10 PBMCs compared with other patients (95% CI: 1.24 to 3.80, P = 0.007). The only other factor associated with progression was follow-up CD4 count (hazard ratio = 1.23 per 100 cells/mm decrease; 95% CI: 1.07 to 1.41, P = 0.003). DISCUSSION:: PBMC HIV-1 DNA level was strongly associated with HIV-1 disease progression, even after adjusting for HIV-1 RNA and CD4 T-cell count. Further studies should assess whether patients with high HIV-1 DNA levels should start antiretroviral therapy earlier than other patients.

Unité(s) : Laboratoire de Microbiologie

Impact of adverse events on outcomes in intensive care unit patients

ORGEAS MG, TIMSIT JF, SOUFIR L, TAFFLET M, ADRIE C, PHILIPPART F, ZAHAR JR, CLEC'H C, GOLDRAN-TOLEDANO D, JAMALI S, DUMENIL AS, AZOULAY E, CARLET J

2008 - Crit. Care Med. 36(7):2041-2047

OBJECTIVE: To examine the association between predefined adverse events (AE) (including nosocomial infections) and intensive care unit (ICU) mortality, controlling for multiple adverse events in the same patient and confounding variables. DESIGN: Prospective observational cohort study of the French OUTCOMEREA multicenter database. SETTING: Twelve medical or surgical ICUs. PATIENTS: Unselected patients hospitalized for > or = 48 hrs enrolled between 1997 and 2003. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of the 3,611 patients included, 1415 (39.2%) experienced one or more AEs and 821 (22.7%) had two or more AEs. Mean number of AEs per patient was 2.8 (range, 1-26). Six AEs were associated with death: primary or catheter-related bloodstream infection (BSI) (odds ratio [OR], 2.92; 95% confidence interval [CI], 1.6-5.32), BSI from other sources (OR, 5.7; 95% CI, 2.66-12.05), nonbacteremic pneumonia (OR, 1.69; 95% CI, 1.17-2.44), deep and organ/space surgical site infection without BSI (OR, 3; 95% CI, 1.3-6.8), pneumothorax (OR, 3.1; 95% CI, 1.5-6.3), and gastrointestinal bleeding (OR, 2.6; 95% CI, 1.4-4.9). The results were not changed when the analysis was confined to patients with mechanical ventilation on day 1, intermediate severity of illness (Simplified Acute Physiology Score II between 35 and 55), no treatment-limitation decisions, or no cardiac arrest in the ICU. CONCLUSIONS: AEs were common and often occurred in combination in individual patients. Several AEs independently contributed to death. Creating a safe ICU environment is a challenging task that deserves careful attention from ICU physicians.

Unité(s) : Laboratoire de Microbiologie

Genetic diversity and phylogeographic distribution of SIV : how to understand the origin of HIV

PEETERS M, CHAIX ML, DELAPORTE E

2008 - M S-Méd. Sci. 24(6-7):621-628

Emergence of human immunodeficiency viruses HIV-1 and HIV-2 results from interspecies transmission from simian viruses SIV. SIVcpzPtt infecting chimpanzees, and from which the HIV-1 (subgroups M and N) is derived is still found in the Pan troglodytes troglodytes population of south Cameroon chimpanzees. The ancestor of HIV-1 group O, is found in the Gorilla residing in Western Africa, but chimpanzees are in fact the initial reservoir of the SIV viruses SIVgor, and it is still unclear whether the group O HIV-1 has been trasmitted to humans by gorillas and/or chimpanzees. At least eight interspecies transmissions between and humans implicating SIVsmm (from sooty mangabey monkeys) have occured, corresponding to the eight VIH-2 groups. Since habits of hunting and meat preparation in the bush still persistently expose humans in Africa to SIV infection, new interspecies transmission of these viruses remains a possibility. double dagger.

Unité(s) : Laboratoire de Microbiologie

Candidiasis emerging species under antibiotic treatment: clinical impact

RIBADEAU-DUMAS F, BOUGNOUX ME, ZAHAR JR, LORTHOLARY O

2008 - M S-Méd. Sci. 24(Sp. Iss. 3):24-31

Candida sp. is the most prevalent fungal pathogen implicated in invasive mycoses. The incidence of such infections, mostly of nosocomial origin, depends on the type of hospitals investigated with approximately 0,3 per thousand admissions in Europe and a 40 % mortality rate at day 30. Among the classical risk factors of invasive candidiasis, antibacterial therapy plays a key role. In the present paper, we will review the influence of antibiotics on the occurrence of Candida colonization and invasive candidiasis in experimental animals and in humans. Antibacterials kill part of the commensal flora, mostly anaerobes and thus enable intestinal yeasts such as Candida sp. to proliferate, adhere to the mucosal surface and spread into the blood. Colonization has been shown to be related to the number of antibacterials administered and their duration of prescription. In addition, the magnitude of mucosal colonization has been correlated with the subsequent occurrence of invasive candidiasis. Among different antibacterial drugs, those with a broad spectrum or impacting commensal flora such as beta-lactams with beta-lactams inhibitors and 3(rd) generation cephalosporins with a marked digestive diffusion, recent fluoroquinolones or ertapenem and vancomycin are associated with an increased rate of yeast colonisation compared to other drugs. Other drugs such as roxithromycin, linezolid, cefepime or aztreonam which exhibit a reduced impact on intestinal flora are less responsible for the occurrence of Candida colonization. The choice of antibacterials in the clinics, its duration of prescription as well as the number of drugs prescribed influence the occurrence of Candida colonization and invasive candidiasis.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

Frequent Occurrence of Chronic Hepatitis B Virus Infection among West African HIV Type-1-Infected Children

ROUET F, CHAIX ML, INWOLEY A, ANAKY MF, FASSINOU P, KPOZEHOUEN A, ROUZIOUX C, BLANCHE S, MSELLATI P

2008 - Clin. Infect. Dis. 46(3):361-366

Background. @nbsp; The aim of this study, conducted in Ivory Coast, was to evaluate the prevalence and evolution of viral hepatitis in children coinfected with human immunodeficiency virus type 1 (HIV-1). Methods. @nbsp; Hepatitis B virus (HBV) and hepatitis C virus (HCV) markers were retrospectively and longitudinally assessed among 280 HIV-1-infected children enrolled in the Agence Nationale de Recherches sur le SIDA et les Hepatites Virales B et C 1244/1278 cohort. Among these, 173 (61.8%) received highly active antiretroviral therapy (HAART), including lamivudine (3TC) for 122 children. Detection of the hepatitis B s antigen (HBsAg) was performed on specimens collected at inclusion and 6 months later. If results of both tests were positive, hepatitis B e antigen (HBeAg)/hepatitis B e antibody (HBeAb) and HBV DNA levels were measured at inclusion and during follow-up. A fourth-generation HCV enzyme immunoassay was used for HCV screening at inclusion. Results. @nbsp; In our pediatric cohort, no patients were infected with HCV, but the prevalence of HBsAg at inclusion was 12.1% (34 of 280; 95% confidence interval [CI], 8.6-16.6). Among the HBV-HIV-1-coinfected children, a high rate of positive HBeAg chronic hepatitis B (CHB) was noted at inclusion (82.4% [ 28 of 34]; 95% CI, 65.5%-93.2%) and after a median follow-up of 18 months (78.3%; 95% CI, 45.5%-92.7%), with no significant difference between children treated with HAART (with or without 3TC) and untreated ones. These children showed high HBV DNA levels (usually >8.0 log(10) copies/mL) and viral population consisting of nearly exclusively wild-type HBeAg-positive HBV strains, strongly suggesting that most of them were in the initial immunotolerant phase of chronic hepatitis B. Conclusion. @nbsp; In sub-Saharan Africa, children with chronic hepatitis B and who are treated with 3TC-based HAART are at risk of developing 3TC resistance. Further studies are required to guide the management of HBV-HIV-1-coinfected children.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie

In-house HIV-1 RNA real-time RT-PCR assays: principle, available tests and usefulness in developing countries

ROUET F, MENAN H, VILJOEN J, NGO-GIANG-HUONG N, MANDALIYA K, VALEA D, LIEN TX, DANAVIAH S, ROUSSET D, GANON A, NERRIENET E

2008 - Expert Rev. Mol. Diagn. 8(5):635-650

The principle of currently available licensed HIV-1 RNA assays is based on real-time technologies that continuously monitor the fluorescence emitted by the amplification products. Besides these assays, in-house quantitative (q) real-time reverse transcription (RT)-PCR (RT-qPCR) tests have been developed and evaluated particularly in developing countries, for two main reasons. First, affordable and generalized access to HIV-1 RNA viral load is urgently needed in the context of expected universal access to prevention and antiretroviral treatment programs in these settings. Second, since many non-B subtypes, circulating recombinant forms and unique recombinant forms circulate in these areas, in-house HIV-1 RNA RT-qPCR assays are ideal academic tools to thoroughly evaluate the impact of HIV-1 genetic diversity on the accuracy of HIV-1 RNA quantification, as compared with licensed techniques. To date, at least 15 distinct in-house assays have been designed. They differ by their chemistry and the HIV-1 target sequence (located in gag, Pol-IN or LTR gene). Analytical performances of the tests that have been extensively evaluated appear at least as good as (or even better than) those of approved assays, with regard to HIV-1 strain diversity. Their clinical usefulness has been clearly demonstrated for early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficacy. The LTR-based HIV-1 RNA RT-qPCR assay has been evaluated by several groups under the auspices of the Agence Nationale de Recherches sur le SIDA et les hepatites virales B et C. It exists now as a complete standardized commercial test.

Unité(s) : Laboratoire de Microbiologie

Prolonged valproic acid treatment does not reduce the size of latent HIV reservoir

SAGOT-LEROLLE N, LAMINE A, CHAIX ML, BOUFASSA F, ABOULKER JP, COSTAGLIOLA D, GOUJARD C, PALLER C, DELFRAISSY JF, LAMBOTTE O

2008 - AIDS 22(10):1125-1129

OBJECTIVE: To investigate the impact of prolonged valproic acid treatment on the HIV reservoir in patients on highly active antiretroviral therapy. DESIGN: In a single-center pilot study, the size of the HIV reservoir of 11 patients receiving valproic acid for seizures for more than 2 years was compared with 13 matched patients. In addition, the outcome of patients receiving valproic acid in the French clinical trials of scheduled treatment interruption was recorded. METHODS: Total and integrated HIV-1 DNA in, respectively, peripheral blood mononuclear cells and CD4 T cells of the patients were quantified by real-time PCR methods. The frequency of CD4 T cells carrying replication-competent virus was estimated by a quantitative limiting-dilution assay in which virus growth was detected by RT-PCR in culture supernatants of activated CD4 T cells.Clinical charts of the patients included in scheduled treatment interruption trials receiving valproic acid were reviewed. RESULTS: Total and integrated HIV DNA were logarithmically more abundant than cells carrying replication-competent virus, but there was no significant difference in these three parameters between the two groups of matched patients.Three patients receiving valproic acid were included in scheduled treatment interruption trials. The rebound of viral replication was similar to that of the other patients of the trials. CONCLUSION: Long-term valproic acid therapy seems to be insufficient to reduce the size of the HIV-1 reservoir.

Unité(s) : Laboratoire de Microbiologie, EA3620

Ritonavir-Boosted Protease Inhibitor Monotherapy for the Treatment of HIV-1 Infection

SAHALI S, CHAIX ML, DELFRAISSY JF, GHOSN J

2008 - AIDS Rev. 10(1):4-14

Guidelines for the use of antiretrovirals for HIV-1 infection recommend combining at least three agents. Toxicities, cost, and the complexity of such regimens warrant the search for other options. Boosted protease inhibitor monotherapy is one of the appealing options being investigated. Herein we review uncontrolled and controlled clinical trials evaluating boosted protease inhibitor monotherapy in several clinical settings: maintenance therapy, induction-maintenance strategies, and first-line treatment. Boosted lopinavir monotherapy has been largely investigated in maintenance and induction-maintenance strategies, showing its ability to maintain viral suppression in the majority of participants. The major concern is the higher proportion of patients experiencing transient episodes of low-level viremia (HIV-RNA 50-500 copies/ml) when compared to classical triple regimens. No protease inhibitor-associated resistance mutation was detected in patients who failed on boosted lopinavir monotherapy. Three uncontrolled maintenance strategy studies with boosted atazanavir monotherapy showed conflicting results. Thus, the reassuring results obtained with lopinavir might not be extended to the whole protease inhibitor class, warranting further studies with new generation protease inhibitors such as darunavir. Finally, one controlled trial comparing first-line boosted lopinavir monotherapy to a standard triple combination showed that the latter outperformed the boosted protease inhibitor monotherapy in this clinical setting. In summary, a boosted protease inhibitor single-agent strategy can maintain continuous plasma HIV-RNA suppression in a large proportion of patients already suppressed on a standard triple combination. The more frequent occurrence of low-level viremia, however, does not allow the widespread use of such a strategy outside of clinical studies at this time.

Unité(s) : Laboratoire de Microbiologie, EA3620

Splenic rupture and malignant mediterranean spotted Fever

SCHMULEWITZ L, MOUMILE K, PATEY-MARIAUD DE SERRE N, POIREE S, GOUIN E, MECHAI F, COCARD V, MAMZER-BRUNEEL MF, ABACHIN E, BERCHE P, LORTHOLARY O, LECUIT M

2008 - Emerg. Infect. Dis. 14(6):995-997

To the Editor: Mediterranean spotted fever (MSF) is a Rickettsia conorii infection endemic to the Mediterranean. In this case, a 55-year-old man was referred to the Necker-Enfants Malades Hospital, Paris, France, for fever, myalgia, and hypotensive shock. The patient had been in Southern France (Montpellier) 6 days before symptom onset and had been bitten by a tick on the left hand. Four days later, he reported fatigue, fever (39 degrees C), and myalgia. His medical history showed polycystic kidney disease, which had necessitated hemodialysis and a kidney transplant. He was receiving ongoing treatment with an immunosuppressive regime of cyclosporine, prednisolone, and tacrolimus; his baseline hemoglobin level was 15 g/dL, and creatinine level was 230 mumol/L.

Unité(s) : Infectiologie, Laboratoire de Microbiologie, Transplantation Adulte

Rapid CD4+ Cell Decrease After Transient cART Initiated During Primary HIV Infection (ANRS PRIMO and SEROCO Cohorts

SENG R, GOUJARD C, DESQUILBET L, SINET M, ROUZIOUX C, DEVEAU C, BOUFASSA F, DELFRAISSY JF, MEYER L, VENET A

2008 - JAIDS 49(3):251-258

OBJECTIVE:: To modelize the rate of CD4 cell count decline and its determinants after cessation of combination antiretroviral therapy (cART) started during primary HIV infection (PHI) and compare it with never-treated patients. METHODS:: Kinetics of CD4 counts were analyzed on the square root scale by using a mixed-effects model in 170 patients who received cART during PHI from the Primary Infection (PRIMO) cohort and 123 never-treated patients from the Seroconverters (SEROCO) cohort. RESULTS:: After cART interruption in the PRIMO cohort, the CD4 cell count fell rapidly during the first 5 months and more slowly thereafter. The timing of treatment initiation had no influence on the rate of CD4 cell decline. In contrast, a larger increase in CD4 cell counts during cART was associated with a steeper decline and a larger loss of CD4 cells after treatment interruption. The mean CD4 cell loss 3 years postinterruption was 383 cells per microliter. In the SEROCO cohort, the CD4 T-cell decline was less steep (3-year CD4 loss 239 cells/muL). As a result, the mean CD4 cell counts were similar (416 cells/muL) 3 years after cART interruption (PRIMO) or after infection (SEROCO). CONCLUSIONS:: These data question the benefit of a limited course of cART even when initiated within 3 months after PHI diagnosis.

Unité(s) : Laboratoire de Microbiologie, EA3620

DETECTION OF CIRCULATING ASPERGILLUS FUMIGATUS DNA BY RT PCR ASSAY ON LARGE SERUM VOLUMES IMPROVES EARLY DIAGNOSIS OF INVASIVE ASPERGILLOSIS IN HIGH-RISK ADULT HAEMATOLOGY PATIENTS

SUAREZ F, LORTHOLARY O, BULAND S, RUBIO MT, GHEZ D, MAHE V, QUESNE G, POIREE S, BUZYN A, VARET B, BERCHE P, BOUGNOUX ME

2008 - J. Clin. Microbiol. 46(11):3772-3777

Galactomannan antigen (GMA) detection in serum is the standard assay for diagnosis of invasive aspergillosis (IA) in high-risk haematology patients. Detection of Aspergillus DNA in serum has been proposed but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. We compared, in a 13-month prospective study, the performances of screening serum for GMA twice-weekly, using an enzyme immunoassay, or weekly for Aspergillus fumigatus DNA, using the real-time polymerase chain reaction (RT-PCR) on 1.0 mL (LSV), or 100 microL (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC/MSG documented IA. In all, 1,870 samples were screened for GMA. Sensitivity (Se), specificity (Sp), positive (PPV) and negative predictive values (NPV) of GMA for IA cases were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 939 samples for DNA extracted from LSV, of which 404 were also tested by using SSV. Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100% respectively, using LSV, and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, using SSV. DNA detection was positive when performed on LSV in 2 IA cases where GM remained negative. Furthermore, in 4 IA cases DNA detection occurred prior to the detection of GMA. The use of LSV for extraction improved the performances of the RT-PCR which appears highly sensitive and specific for the early diagnosis of IA in high-risk haematology patients.

Unité(s) : Anesthésie Réanimation, Hématologie Adulte, Infectiologie, Laboratoire de Microbiologie, Radiologie Adulte, SAMU - SMUR

Characterization of an Old Complex Circulating Recombinant Form, CRF27_cpx, Originating from the Democratic Republic of Congo (DRC) and Circulating in France

VIDAL N, FRANGE P, CHAIX ML, MULANGA C, LEPIRA F, BAZEPEO SE, GOUJARD C, MEYER L, ROUZIOUX C, DELAPORTE E, PEETERS M

2008 - AIDS Res. Hum. Retroviruses 24(2):315-321

ABSTRACT Full-length genomes were characterized for two samples, 02CD-LBR024 from the Democratic Republic of Congo (DRC) and 04FR-CD-KZS from France, that formed a separate subcluster with a previously characterized env subtype E isolate from DRC with a recombinant structure different from CRF01-AE. Since the three viruses are clearly epidemiologically unlinked and share the same complex recombinant structure, they represent a circulating recombinant form, designated as CRF27-cpx. The recombination pattern involves six different HIV-1 subtypes (A, E, G, H, J, and K) and a small unclassified fragment. The genetic distances are relatively high, indicating that CRF27-cpx evolved over a long time. Their prevalence is low (0.75%) and remained stable over time in the DRC. The existence of the 04FR.CD.KZS virus, in a patient who recently seroconverted in France, confirmed that these strains now circulate outside the DRC. Continuous monitoring of HIV-1 strains thus remains important to allow early identification of the introduction of new variants.

Unité(s) : Laboratoire de Microbiologie, EA3620

Mother-to-child HIV transmission despite antiretroviral therapy in the ANRS French Perinatal Cohort

WARSZAWSKI J, TUBIANA R, LE CHENADEC J, BLANCHE S, TEGLAS JP, DOLLFUS C, FAYE A, BURGARD M, ROUZIOUX C, MANDELBROT L

2008 - AIDS 22(2):289-299

OBJECTIVE:: To identify factors associated with mother-to-child HIV-1 transmission (MTCT) from mothers receiving antenatal antiretroviral therapy. DESIGN:: The French Perinatal Cohort (EPF), a multicenter prospective cohort of HIV-infected pregnant women and their children. METHODS:: Univariate analysis and logistic regression, with child HIV status as dependent variable, were conducted among 5271 mothers who received antiretroviral therapy during pregnancy, delivered between 1997 and 2004 and did not breastfeed. RESULTS:: The MTCT rate was 1.3% [67/5271; 95% confidence interval (CI), 1.0-1.6]. It was as low as 0.4% (5/1338; 95% CI, 0.1-0.9) in term births with maternal HIV-1 RNA level at delivery below 50 copies/ml. MTCT increased with viral load, short duration of antiretroviral therapy, female gender and severe premature delivery: 6.6% before 33 weeks versus 1.2% at 37 weeks or more (P < 0.001). The type of antiretroviral therapy was not associated with transmission. Intrapartum therapy was associated with four-fold lower MTCT (P = 0.04) in case of virological failure (> 10 000 copies/ml). Elective cesarean section tended to be inversely associated with MTCT in the overall population, but not in mothers who delivered at term with viral load < 400 copies/ml [odds ratio (OR), 0.83; 95% CI, 0.29-2.39; P = 0.37]. Among them, only duration of antenatal therapy was associated with transmission (OR by week, 0.94; 95% CI, 0.90-0.99; P = 0.03). CONCLUSIONS:: Low maternal plasma viral load is the key factor for preventing MTCT. Benefits in terms of MTCT reduction may be expected from early antiretroviral prophylaxis. The potential toxicity of prolonged antiretroviral use in pregnancy should be evaluated.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie, EA3620

Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover

ALMEIDA JR, PRICE DA, PAPAGNO L, ARKOUB ZA, SAUCE D, BORNSTEIN E, ASHER TE, SAMRI A, SCHNURIGER A, THEODOROU I, COSTAGLIOLA D, ROUZIOUX C, AGUT H, MARCELIN AG, DOUEK D, AUTRAN B, APPAY V

2007 - J. Exp. Med. 204(10):2473-2485

The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)-B27 patients. To understand further the nature of CD8+ T cell-mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27-restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10-specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines.

Unité(s) : Laboratoire de Microbiologie

Prevalence of resistance to nevirapine in mothers and children after single-dose exposure to prevent vertical transmission of HIV-1: a meta-analysis

ARRIVÉ E, NEWELL ML, EKOUEVI DK, CHAIX ML, THIEBAUT R, MASQUELIER B, LEROY V, PERRE PV, ROUZIOUX C, DABIS F

2007 - Int. J. Epidemiol. 36(5):1009-1021

BACKGROUND: Single-dose nevirapine (NVP) is the main option for the prevention of mother-to-child transmission (PMTCT) of HIV-1 in countries with limited resources. However, the use of single-dose NVP results in HIV-1 viral resistance which could compromise the success of subsequent treatment of mother and child with antiretroviral combinations that include non-nucleosidic-reverse-transcriptase inhibitors. This systematic review and meta-analysis of summarized data aimed to estimate the proportion of mothers and children with NVP resistance mutations detected in plasma samples 4-8 weeks postpartum after single-dose NVP use for PMTCT. METHODS: Systematic search of electronic databases (MEDLINE, PASCAL) and conference proceedings (1997 to February 2006). Inclusion of all studies, without design, place or language restrictions, meeting the following criteria: use of single-dose NVP; viral genotyping performed with standard sequence analyses, between 4 and 8 weeks postpartum, in plasma samples; available public report; report of mothers' median baseline plasma HIV-1 RNA levels. Data extraction by two independent reviewers using a standardized form created for this purpose. Logistic random effect models to obtain pooled estimates. Univariable and multivariable meta-regression to explore sources of heterogeneity. RESULTS: The pooled estimate of NVP resistance prevalence was 35.7% [95% confidence interval (CI) 23.0-50.6] in women in 10 study arms using single-dose NVP +/- other antepartum antiretrovirals and 4.5% (CI 2.1-9.4) in three study arms providing also postpartum antiretrovirals (adjusted odds ratio 0.08; CI 0.04-0.16). The corresponding estimates in children were 52.6% (CI 37.7-67.0) in seven study arms using single-dose NVP only and 16.5% (CI 8.9-28.3) in eight study arms combining single-dose NVP with other antiretrovirals. CONCLUSIONS: Single-dose NVP is widely used for PMTCT in resource-poor settings, but the burden of viral resistance is high in both women and children. It is substantially lower in studies providing additional postpartum antiretrovirals. The clinical implications of these findings should be further investigated.

Unité(s) : Laboratoire de Microbiologie

Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior

AUFFRAY C, FOGG D, GARFA M, ELAIN G, JOIN-LAMBERT O, KAYAL S, SARNACKI S, CUMANO A, LAUVAU G, GEISSMANN F

2007 - Science 317(5838):666-670

The cellular immune response to tissue damage and infection requires the recruitment of blood leukocytes. This process is mediated through a classical multistep mechanism, which involves transient rolling on the endothelium and recognition of inflammation followed by extravasation. We have shown, by direct examination of blood monocyte functions in vivo, that a subset of monocytes patrols healthy tissues through long-range crawling on the resting endothelium. This patrolling behavior depended on the integrin LFA-1 and the chemokine receptor CX(3)CR1 and was required for rapid tissue invasion at the site of an infection by this "resident" monocyte population, which initiated an early immune response and differentiated into macrophages.

Unité(s) : Chirurgie Viscérale Pédiatrique, Laboratoire de Microbiologie, U838, IRNEM

Failure of bone marrow transplantation to eradicate HIV reservoir despite efficient HAART

AVETTAND-FENOEL V, MAHLAOUI N, CHAIX ML, MILLIANCOURT C, BURGARD M, CAVAZZANA-CALVO M, ROUZIOUX C, BLANCHE S

2007 - AIDS 21(6):776-777

Unité(s) : Biothérapie, Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie

Une histoire des microbes

BERCHE P

2007 - Ouvrage

« Vers trois heures de l'après-midi, je vis des petites anguilles ou des petits vers qui s'enchevêtraient et se tortillaient. C'était parmi toutes les merveilles de la nature, la plus prodigieuse, et je dois ajouter que je n'ai pas éprouvé de plus grand plaisir que celui de voir plusieurs milliers de créatures dans une gouttelette d'eau se déplacer les unes parmi les autres, chacune animée de son propre mouvement. ». Dans une infusion de poire, avec un microscope rudimentaire, Antoine van Leeuwenhoek, obscur drapier de Delft, fut le premier être humain à observer des bactéries. Cette incroyable découverte date du 10 juillet 1676, et pourtant, les microbes furent les premiers créatures vivantes apparues sur Terre il y a plus de trois milliards d'années. D'êtres unicellulaires, les microbes ont évolué vers des organismes complexes qui ont donné naissance aux plantes, aux animaux et... aux hommes. A l'heure où de nouvelles menaces liées à l'émergence de nouveaux microbes, de nouvelles pandémies et du bioterrorisme, peuvent mettre en péril notre survie, cet ouvrage érudit et vivant est un plaidoyer montrant que nous n'avons jamais eu autant besoin d'une science maîtrisée au service du progrès humain..

Unité(s) : Laboratoire de Microbiologie

Rapid identification of Staphylococci isolated in clinical microbiology laboratories by Matrix Assisted Laser Desorption Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS)

CARBONNELLE E, BERETTI JL, COTTYN S, QUESNE G, BERCHE P, NASSIF X, FERRONI A

2007 - J. Clin. Microbiol. 45(7):2156-2161

Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) of intact bacteria yield reproducible spectrum depending upon growth conditions, strain or species. Using whole viable bacteria we describe here the application of MALDI-TOF-MS to the identification of coagulase negative Staphylococci (CNS). Our aim was, once a bacterium has been recognized as Micrococcaceae, to identify peaks, in the spectrum, that can be used to identify the species or subspecies. MALDI-TOF-MS was performed using bacteria obtained from one isolated colony. One reference strain for each of the 23 clinically relevant species or subspecies of Micrococcaceae was selected. For each reference strain, the MALDI-TOF-MS profile of 10 colonies obtained from 10 different passages was analyzed. For each strain, only peaks that were conserved in the spectra of all 10 isolated colonies and with a relative intensity above 0.1 were retained, thus leading to a set of 3 to 14 selected peaks per strain. The MALDI-TOF-MS profile of 196 tested strains was then compared with that of the set of selected peaks of each of the 23 reference strains. In all cases the best hit was with the set of peaks of the reference strain belonging to the same species as that of the tested strain, thus demonstrating that the 23 sets of selected peaks can be used as a database for the rapid species identification of CNS. Similar results were obtained using 4 different growth conditions. Extending this strategy to other groups of relevant pathogenic bacteria will allow rapid bacterial identification.

Unité(s) : Laboratoire de Microbiologie

Response to HAART in French patients with resistant HIV-1 treated at primary infection: ANRS Resistance Network

CHAIX ML, DESQUILBET L, DESCAMPS D, COSTAGLIOLA D, DEVEAU C, GALIMAND J, GOUJARD C, SIGNORI-SCHMUCK A, SCHNEIDER V, TAMALET C, PELLEGRIN I, WIRDEN M, MASQUELIER B, BRUN-VEZINET F, ROUZIOUX C, MEYER L, FRANCH PRIMO COHORT STUDY GROUP

2007 - Antivir. Ther. 12(8):1305-1310

Objective: The aim of the study was to analyse the response to highly active antiretroviral therapy (HAART) initiated at the time of primary HIV infection (PHI) in patients infected with a virus resistant to >= 1 drug of their treatment compared with patients infected with a wild-type virus. Methods: We analysed data from 350 patients who were enrolled from 1996-2004 in the French ANRS PRIMO Cohort or in the ANRS Resistance Group and treated with HAART during PHI. During the study period, HAART was initiated before the result of the genotypic resistance test was available. We compared patients infected with a virus resistant to A drug of their regimen (GR group, n=46) with patients harbouring a wild-type virus (WT group, n=304). Virological and immunological response to treatment according to drug-resistance profile was analysed 3 months and 6 months after HAART initiation. Results: In GR and WT groups, HIV RNA level was <400 copies/ml in 68% and 83% (P=0.02) and <50 copies/ml in 23% and 40% (P=0.08) 3 months after HAART initiation. In multivariable logistic regression taking into account gender, age, boosted PI regimen, plasma HIV RNA and CD4(+) T-cell count at HAART initiation, patients with virus resistant to A drug of their regimen were significantly less likely to achieve undetectable viral load at month 3 (odds ratio 0.32, 95% confidence interval 0.15-0.72) than the others. This difference was sustained up to month 6. Conclusion: In this large cohort of HAART-treated PHI-patients, the presence of drug resistance mutations led to suboptimal response to early therapy.

Unité(s) : Laboratoire de Microbiologie, EA3620

Impact of Nevirapine (NVP) Plasma Concentration on Selection of Resistant Virus in Mothers Who Received Single-Dose NVP To Prevent Perinatal Human Immunodeficiency Virus Type 1 Transmission and Persistence of Resistant Virus in Their Infected Childre

CHAIX ML, EKOUEVI DK, PEYTAVIN G, ROUET F, TONWE-GOLD B, VIHO I, BEQUET L, AMANI-BOSSE C, MENAN H, LEROY V, ROUZIOUX C, DABIS F

2007 - Antimicrob. Agents Chemother. 51(3):896-901

Nonnucleoside reverse transcriptase inhibitor resistance following the use of single-dose nevirapine (sdNVP) for the prevention of mother-to-child transmission (PMTCT) remains a concern. In the ANRS-1201/1202 Ditrame study, conducted in Abidjan, Cote d'Ivoire, a short-course regimen of zidovudine was associated with sdNVP for PMTCT. In this study, we estimate the frequency of NVP resistance and its relationship with NVP concentration in mothers. Genotypic resistance analysis was performed on mothers' plasma samples at week 4 postpartum (PP) and on human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells (PBMC) when an NVP resistance mutation was detected. The same tests were performed for the infected children at week 4, month 3, and month 12. Mothers' NVP plasma concentrations were measured at 48 h PP. Twenty-one (33%) of the 63 women selected had NVP-resistant (NVP-R) virus at week 4 PP. The median plasma NVP concentration was 598 ng/ml for the mothers without NVP-R virus compared to 851 ng/ml for the mothers harboring NVP-R virus (P = 0.014). NVP-R mutations were detected in the HIV DNA of 15/20 women. Plasma NVP-R mutations were detectable in 6 of 26 infected children at week 4. All 6 children had detectable NVP-R mutations in HIV DNA of PBMC. Blood samples taken at month 3 (1 child) and month 12 (1 child) revealed the persistence of NVP-R mutations in plasma and cells. Emergence of NVP-R virus in mothers is strongly correlated with a high level of plasma NVP concentration, owing to a prolonged postpartum period of viral replication under NVP selective pressure. The follow-up of the cohort demonstrates the prolonged archive of resistant virus.

Unité(s) : Laboratoire de Microbiologie, EA3620

Alternative Neisseria spp. type IV pilin glycosylation with a glyceramido acetamido trideoxyhexose residue

CHAMOT-ROOKE J, ROUSSEAU B, LANTERNIER F, MIKATY G, MAIREY E, MALOSSE C, BOUCHOUX G, PELICIC V, CAMOIN L, NASSIF X, DUMENIL G

2007 - Proc. Nat. Acad. Sci. USA 104(37):14783-14788

The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pili. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido-2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by "top-down" MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pili preparations originating from N. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis.

Unité(s) : Laboratoire de Microbiologie, U570

Contribution of molecular tools for the diagnosis of endogenous pneumococcal endophthalmitis in a patient with the acquired immunodeficiency syndrome

CHANDESRIS MO, HOT A, JOIN-LAMBERT O, LOULERGUE P, FERRONI A, LORTHOLARY O, VIARD JP

2007 - Scand. J. Infect. Dis. 39(11):1080-1081

Confirmation of infectious endophthalmitis is a major challenge. Traditional microbial tools are frequently unsuccessful mostly due to very small fluid samples and prior use of antibiotics. Here we report the first case of Streptococcus pneumoniae endophthalmitis diagnosed by polymerase chain reaction. Molecular biology should be considered as a new tool for the diagnosis of such sequestered sites as the eye.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

PROPIONIBACTERIUM ACNES ENDOCARDITIS IN AN ADOLESCENT BOY SUFFERING FROM A CONGENITAL CARDIOPATHY

CHARLES P, HOT A, OU P, CARBONNELLE E, SIDI D, NASSIF X, LORTHOLARY O

2007 - Pediat. Inf. Dis. J. 26(9):856-858

Propionibacterium acnes endocarditis is an uncommon infection in pediatrics. We describe a case of P. acnes endocarditis in a 16-year-old boy that occurred 6 months after recurrent surgery for a congenital cardiopathy. Molecular identification of P. acnes was obtained. He recovered from this infection after a surgical treatment and with a prolonged antibiotic regimen including ceftriaxone.

Unité(s) : Cardiologie Pédiatrique, Infectiologie, Laboratoire de Microbiologie

Shorten treatment of in children severe osteomyelitis

COHEN R

2007 - Archives Pédiatrie 14(Suppl. 2):S128-S130

Recommendations on treatment of acute staphylococcal osteomyelitis of children, based mostly on retrospective analyses, comprise up to 6 weeks for antimicrobials and the possibility of switching to the oral route only when the clinical and biological situation are under control. Some prospective non comparative studies suggest a good efficacy of simplified regimen: treatment initiated intravenously but switched to oral administration within few days, using high dosage. However, this type of treatment should be considered only for oral drugs with good pharmacokinetics/pharmacodynamic parameters as clindamycin of some first generation cephalosporins.

Unité(s) : Laboratoire de Microbiologie

Prevalence of HIV-1 Drug Resistance in Treated Patients: A French Nationwide Study

COSTAGLIOLA D, DESCAMPS D, ASSOUMOU L, PH M, MORAND JOUBERT L, MARCELIN AG, BRODARD V, DELAUGERRE C, MACKIEWICZ V, RUFFAULT A, IZOPET J, PLANTIER JC, TAMALET C, YERLY S, SAIDI S, BRUN VEZINET F, MASQUELIER B

2007 - JAIDS 46(1):12-18

BACKGROUND:: Surveillance of HIV-1 drug resistance in antiretroviral-treated patients is important from the public health perspective of the spread of resistance and to evaluate the proportion of patients for whom new drugs are needed. METHODS:: Patients were consecutively included in 28 centers in France and 1 center in Switzerland if they had a viral load measurement performed in June 2004, with a result >/=1000 copies/mL. Reverse transcriptase, protease, and gp41 genes were sequenced, and resistance mutations were reported as listed on the Web site (www.iasusa.org). The genotypic resistance results were interpreted by the Agence Nationale de Recherches sur le SIDA et les Hepatites Virales (ANRS) and Stanford algorithms. RESULTS:: The 498 patients included had been exposed to 9 (interquartile ratio [IQR]: 6 to 12) antiretroviral drugs. Patients' viruses harbored 4 nucleoside reverse transcriptase inhibitor (IQR: 1 to 6) and 4 protease inhibitor (PI; IQR: 2 to 8) resistance mutations, whereas 44% had at least 1 nonnucleoside reverse transcriptase inhibitor resistance mutation. The frequency of resistance to at least 1 drug was 88% with the ANRS algorithm and 83% with the Stanford algorithm. The frequencies of complete resistance to 1, 2, and 3 classes of drugs were 37%, 15%, and 4%, respectively, with the ANRS algorithm and 27%, 23%, and 24%, respectively, with the Stanford algorithm. The most important differences between algorithms were for PIs. Using the ANRS algorithm and extrapolation on the whole French database, 19% of all treated patients could contribute to the spread of resistance and 4% had complete resistance to 2 classes of antiretroviral drugs. CONCLUSIONS:: The observed patterns of resistance are linked to a long-lasting history of antiretroviral therapy. The frequency of multiresistance can vary according to the interpretation systems.

Unité(s) : Laboratoire de Microbiologie

HIV-1 co-infection prevalence in two cohorts of early HIV-1 seroconverters in France

COURGNAUD V, SENG R, BECQUART P, BOULAHTOUF A, ROUZIOUX C, BOUFASSA F, DEVEAU C, VAN DE PERRE P, MEYER L, FOULONGNE V

2007 - AIDS 21(8):1055-1056

Despite anecdotal reports of HIV-1 co-infections and super-infections, few large-scale prevalence studies are available on multiple HIV-1 infection. We systematically searched for HIV-1 co-infections by means of a heteroduplex mobility assay in 660 HIV-1 seroconverters from the two ANRS SEROCO and PRIMO cohorts. Our results strongly suggest that HIV-1 co-infection remains a rare phenomenon in HIV-1 seroconverters infected in France between 1986 and 2004.

Unité(s) : Laboratoire de Microbiologie

HIV-1 drug resistance in French infected-children: from newborn to adolescent

DELAUGERRE C, CHAIX ML, WARSZAWSKI J, ROUZIOUX C, BLANCHE S

2007 - Archives Pédiatrie 14(3):298-302

Limit of antiretroviral treatment success is the emergence of drug-resistant virus. As reported in adult population, prevalence of resistance was high in treated HIV-infected children with detectable HIV viral load. Resistance increased with number of prior antiretroviral treatments, particularly with protease inhibitors. Adolescent boys seem at greater risk to harbor multi-classes resistant virus. In HIV-infected newborns, prevalence of resistance was 20%. Most of resistance mutations detected were in accord to perinatal antiretroviral exposition. Principal mechanism of resistance acquisition in newborns was transmission of resistant viruses from mother to child with early archive in cellular reservoir and long term persistence with or without treatment. Consequences of long term therapeutic strategies in children are major.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie

Transmission of multidrug-resistant HIV-1: 5 years of immunological and virological survey

DELAUGERRE C, MARCELIN AG, SOULIE C, CHAIX ML, KATLAMA C, GIRARD PM, CALVEZ V, MORAND-JOUBERT L

2007 - AIDS 21(10):1365-1367

Multidrug resistant HIV-1 acquired at the time of primary infection in two patients persisted for at least 5 years with and without treatment. In each patient, only one back mutation to wild-type codon in the protease gene occurred, and that was concomitantly associated with a marked CD4 cell count decrease. In both cases, infection was caused by CCR5 viruses and no rapid clinical progression to AIDS after primary infection was observed.

Unité(s) : Laboratoire de Microbiologie

Prevalence and risk factors associated with antiretroviral resistance in HIV-1-infected children

DELAUGERRE C, WARSZAWSKI J, CHAIX ML, VEBER F, MACASSA E, BUSEYNE F, ROUZIOUX C, BLANCHE S

2007 - J. Med. Virol. 79(9):1261-1269

In the USA and West Europe, nearly 80% of HIV-1-infected adults, experiencing virologic failure, harbored virus strain resistant to at least one antiretroviral drug. Limited data are available on antiretroviral drug resistance in pediatric HIV infection. The aims of this study were to analyze prevalence of HIV-1 drug resistance and to identify risk factors associated with resistance in this population. Prevalence of genotypic resistance was estimated retrospectively in treated children who experienced virologic failure (with HIV-1-RNA > 500 copies/ml) followed in Necker hospital between 2001 and 2003. Among 119 children with resistance testing, prevalence of resistance to any drug was 82.4%. Resistance ranged from 76.5% to nucleoside reverse transcriptase inhibitor (NRTI), to 48.7% to non-nucleoside reverse transcriptase inhibitor (NNRTI) and 42.9% to protease inhibitor (PI). Resistance to at least one drug of two classes and three classes (triple resistance) was 31.9 and 26.9%, respectively. Resistance was not associated with geographic origin, HIV-1 subtype, and CDC status. In multivariate analysis, resistance to any drug remained associated independently with current low viral load and high lifetime number of past PI. Triple resistance was independently associated with the high lifetime number of past PI and with gender, particularly among children aged 11 years old or more with a prevalence seven times higher in boys than in girls. In conclusion, antiretroviral resistance is common among treated HIV-1-infected children and prevalence was similar with those observed in adult population in the same year period. However, adolescent boys seem to be at greater risk. J. Med. Virol. 79:1261-1269, 2007. (c) Wiley-Liss, Inc.

Unité(s) : Laboratoire de Microbiologie, Immuno-Hématologie-Rhumatologie Pédiatriques

Positive outcomes of HAART at 24 months in HIV-infected patients in Cambodia

FERRADINI L, LAUREILLARD D, PRAK N, NGETH C, FERNANDEZ M, PINOGES L, PUERTAS G, TABURET AM, LY N, ROUZIOUX C, BALKAN S, QUILLET C, DELFRAISSY JF

2007 - AIDS 21(17):2293-2301

Objectives: African and Asian cohort studies have demonstrated the feasibility and efficacy of HAART in resource-poor settings. The long-term virological outcome and clinico-immunological criteria of success remain important questions. We report the outcomes at 24 months of antiretroviral therapy (ART) in patients treated in a Medecins Sans Frontieres/Ministry of Health programme in Cambodia. Methods: Adults who started HAART 24 2 months ago were included. Plasma HIV-RNA levels were assessed by real-time polymerase chain reaction. Factors associated with virological failure were analysed using logistic regression. Results: Of 416 patients, 59.2% were men; the median age was 33.6 years. At baseline, 95.2% were ART naive, 48.9% were at WHO stage IV, and 41.6% had a body mass index less than 18 kg/m(2). The median CD4 cell count was 11 cells/mu l. A stavudine-lamivudine-efavirenz-containing regimen was initiated predominantly (81.0%). At follow-up (median 23.8 months), 350 (84.1%) were still on HAART, 53 (12.7%) had died, six (1.4%) were transferred, and seven (1.7%) were lost to follow-up. Estimates of survival were 85.5% at 24 months. Of 346 tested patients, 259 (74.1 %) had CD4 cell counts greater than 200 cells/mu l and 306 (88.4%) had viral loads of less than 400 copies/ml. Factors associated with virological failure at 24 months were non-antiretroviral naive, an insufficient CD4 cell gain of less than 350 cells/mu l or a low trough plasma ART concentration. In an intention-to-treat analysis, 73.6% of patients were successfully treated. Conclusion: Positive results after 2 years of advanced HIV further demonstrate the efficacy of HAART in the medium term in resource-limited settings. (c) 2007 Wolters Kluwer Health.

Unité(s) : Laboratoire de Microbiologie, EA3620

Epidemiology and bacteriological diagnosis of paediatric acute osteoarticular infections

FERRONI A

2007 - Archives Pédiatrie 14(Suppl. 2):S91-S96

Acute paediatric osteo-articular infections require a fast and sensitive diagnosis allowing a treatment directed to the causative pathogen. Many micro-organisms can be incriminated, but Staphylococcus aureus and Kingella kingae markedly prevail. K. kingae became the first bacterial species responsible for septic arthritis in children < 3 years. More rarely, (2)haemolytic Streptococci and Streptococcus pneumoniae are found. The incidence of community acquired S. aureus resistant to oxacillin in osteo-articular infections is still low in France. The microbiological diagnosis of septic arthritis relies upon analysis of articular fluid, which requires systematic inoculation of a blood culture vial to increase the recovery rate of K. kingae. If the culture is negative, it is recommended to carry out a universal PCR or a PCR targeted to the main germs responsible for septic arthritis. Indeed, PCR represents an undeniable benefice for the diagnosis of paediatric septic arthritis, particularly for the DNA detection of K. kingae. The diagnosis of acute osteomyelitis relies primarily upon blood cultures, since the bone puncture is not a systematic procedure in this setting. Their efficiency is low, and there is still a need to look for other arguments of diagnosis such as search of possible portals of entry or specific serologies.

Unité(s) : Laboratoire de Microbiologie

The HIV RNA setpoint theory revisited

GESKUS RB, PRINS M, HUBERT JB, MIEDEMA F, BERKHOUT B, ROUZIOUX C, DELFRAISSY JF, MEYER L

2007 - Retrovirology 4(.):65

BACKGROUND: The evolution of plasma viral load after HIV infection has been described as reaching a setpoint, only to start rising again shortly before AIDS diagnosis. In contrast, CD4 T-cell count is considered to show a stable decrease. However, characteristics of marker evolution over time depend on the scale that is used to visualize trends. In reconsidering the setpoint theory for HIV RNA, we analyzed the evolution of CD4 T-cell count and HIV-1 RNA level from HIV seroconversion to AIDS diagnosis. Follow-up data were used from two cohort studies among homosexual men (N = 400), restricting to the period before highly active antiretroviral therapy became widely available (1984 until 1996). Individual trajectories of both markers were fitted and averaged, both from seroconversion onwards and in the four years preceding AIDS diagnosis, using a bivariate random effects model. Both markers were evaluated on a scale that is directly related to AIDS risk. RESULTS: Individuals with faster AIDS progression had higher HIV RNA level six months after seroconversion. For CD4 T-cell count, this ordering was less clearly present. However, HIV RNA level and CD4 T-cell count showed qualitatively similar evolution over time after seroconversion, also when stratified by rate of progression to AIDS. In the four years preceding AIDS diagnosis, a non-significant change in HIV RNA increase was seen, whereas a significant biphasic pattern was present for CD4 T-cell decline. CONCLUSION: HIV RNA level has more setpoint behaviour than CD4 T-cell count as far as the level shortly after seroconversion is concerned. However, with respect to the, clinically more relevant, marker evolution over time after seroconversion, a setpoint theory holds as much for CD4 T-cell count as for HIV RNA level.

Unité(s) : Laboratoire de Microbiologie

Interruption of Antiretroviral Therapy Initiated during Primary HIV-1 Infection: Impact of a Therapeutic Vaccination Strategy Combined with Interleukin (IL)-2 Compared with IL-2 Alone in the ANRS 095 Randomized Study

GOUJARD C, MARCELLIN F, HENDEL-CHAVEZ H, BURGARD M, MEIFFREDY V, VENET A, ROUZIOUX C, TAOUFIK Y, EL HABIB R, BEUMONT-MAUVIEL M, ABOULKER JP, LEVY Y, DELFRAISSY JF

2007 - AIDS Res. Hum. Retroviruses 23(9):1105-1113

HIV-specific T cell responses play a critical role in the control of infection. We evaluated the impact of immune-based interventions in patients first treated during primary HIV-1 infection (PHI). Forty-three patients were randomized within three groups, to receive either interleukin-2 (IL-2 group), or boosts of ALVAC-HIV (vCP1433) and LIPO-6T followed by interleukin-2 (Vac-IL2 group), compared with no immune intervention (control group), and were monitored for T cell responses. Impact of strategies on viral replication was subsequently assessed during long-term treatment interruption. HIV-specific CD4(+) T cell responses did not change during the study period in immunized patients relative to controls, and vaccination had only a transient effect on interferon-gamma-producing CD8 responses. Viral rebound after treatment interruption was similar in immunized patients and controls. Forty percent of patients had HIV RNA values <10,000 copies/ml 12 weeks after interruption. The cumulative time off treatment represented almost half the total follow-up period. Immunological and virological status during PHI and HIV DNA load at interruption were predictive of the level of viral rebound after treatment interruption, whereas HIV RNA level during PHI and HIV DNA level at interruption were predictive of the time off treatment. Treatment interruption is safe in patients treated early after primary HIV infection. On the basis of this pilot study, HIV immunizations and interleukin-2 appear to have no supplementary benefit.

Unité(s) : Laboratoire de Microbiologie, EA3620

Pasteurella multocida sepsis and meningitis in 2-month-old twin infants after household exposure to a slaughtered sheep

GUILLET C, JOIN-LAMBERT O, CARBONNELLE E, FERRONI A, VACHÉE A

2007 - Clin. Infect. Dis. 45(6):e80-e81

This article outlines 2 cases of Pasteurella multocida sepsis and meningitis in 2-month-old twins after their father was exposed to a sheep during a sacrifice celebration at home. These cases emphasize the necessity of respecting basic hygiene rules and the danger involved in animal sacrifice without suitable professional structure.

Unité(s) : Laboratoire de Microbiologie

3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili

HELAINE S, DYER DH, NASSIF X, PELICIC V, FOREST KT

2007 - Proc. Nat. Acad. Sci. USA 104(40):15888-15893

Type IV pili (Tfp) are widespread filamentous bacterial organelles that mediate multiple virulence-related phenotypes. They are composed mainly of pilin subunits, which are processed before filament assembly by dedicated prepilin peptidases. Other proteins processed by these peptidases, whose molecular nature and mode of action remain enigmatic, play critical roles in Tfp biology. We have performed a detailed structure/function analysis of one such protein, PilX from Neisseria meningitidis, which is crucial for formation of bacterial aggregates and adhesion to human cells. The x-ray crystal structure of PilX reveals the alpha/beta roll fold shared by all pilins, and we show that this protein colocalizes with Tfp. These observations suggest that PilX is a minor, or low abundance, pilin that assembles within the filaments in a similar way to pilin. Deletion of a PilX distinctive structural element, which is predicted to be exposed on the filament surface, abolishes aggregation and adhesion. Our results support a model in which surface-exposed motifs in PilX subunits stabilize bacterial aggregates against the disruptive force of pilus retraction and illustrate how a minor pilus component can enhance the functional properties of pili of rather simple composition and structure.

Unité(s) : Laboratoire de Microbiologie, U570

Host and bacterial determinants of initial severity and outcome of Escherichia coli sepsis

JAUREGUY F, CARBONNELLE E, BONACORSI S, CLEC'H C, CASASSUS P, BINGEN E, PICARD B, NASSIF X, LORTHOLARY O

2007 - Clin. Microbiol. Infection 13(9):854-862

A 1-year prospective cohort study of all episodes of Escherichia coli bacteraemia in two French university hospitals was conducted to assess simultaneously the influence of host and bacterial determinants on the initial severity and outcome of E. coli sepsis. Clinical data (community-acquired/nosocomial infection, immune status, underlying disease, primary source of infection, severity sepsis scoring and outcome), phylogenetic groups (A, B1, D and B2), nine virulence factors (VFs) (papC, papGII, papGIII, sfa/foc, hlyC, cnf1, iucC, fyuA and iroN) and the antibiotic susceptibility of isolates were investigated. All VFs except iucC were significantly more prevalent (p <0.05) among the B2 group isolates. The non-B2 isolates were more frequently resistant to antibiotics than were B2 isolates (p <0.05). There were significantly more B2 isolates from immunocompetent than from immunocompromised patients (p <0.05). No bacterial or host determinants influenced the initial severity of sepsis. Multivariate analysis revealed that the presence of papGIII, septic shock at baseline and a non-urinary tract origin of sepsis were associated independently with a fatal outcome (p 0.04, <0.0001 and 0.04, respectively). A factorial analysis of correspondence allowed two populations of isolates to be distinguished: those belonging to the B2 group were associated more frequently with susceptibility to antibiotics, community-acquired infection, a urinary tract origin and immunocompetent hosts; those belonging to the A, B1 or D groups were associated more frequently with resistance to antibiotics, a nosocomial origin, a non-urinary tract source and immunocompromised hosts. Although no influence of host or bacterial determinants on the initial severity of sepsis was detected, bacterial and host determinants both influenced the outcome of E. coli sepsis significantly.

Unité(s) : Infectiologie, Laboratoire de Microbiologie, U570

Comparison of cefoxitin and moxalactam 30 microg disc diffusion methods for detection of methicillin resistance in coagulase-negative staphylococci

JOIN-LAMBERT OF, CLAUSER S, GUILLET C, JAIS JP, ABACHIN E, QUESNES G, CARBONNELLE E, LE MONNIER A, ZAHAR JR, KAYAL S, BERCHE P, FERRONI A

2007 - J. Antimicrob. Chemother. 59(4):763-766

OBJECTIVES: To compare cefoxitin and/or moxalactam 30 microg disc diffusion (DD) methods to detect methicillin resistance in coagulase-negative staphylococci (CoNS) using both high- and low-density (HD/LD) inoculum techniques. METHODS: A challenge set of 192 CoNS was tested. DD test results were compared with PBP2a detection. RESULTS: With the LD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 94.4%/100% and 100%/92.4%, respectively, using the DD breakpoints of the Comite de l'Antibiogramme de la Societe Francaise de Microbiologie. With the HD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 93.7%/100% and 100%/96.9%, using the cefoxitin DD breakpoints of the CLSI and a resistant/susceptible breakpoint of < 20 mm/>or=20 mm for moxalactam. Comparison of receiver operating characteristic AUCs did not show significant difference between studied assays, but the overlapping zone where both PBP2a-positive and PBP2a-negative isolates were observed concerned a lower number of strains with moxalactam than with cefoxitin (P < 0.001). Combination of cefoxitin and moxalactam DD methods demonstrated that all isolates with a concordant cefoxitin/moxalactam phenotype were correctly classified. Interestingly, all isolates misclassified by each DD method used alone were cefoxitin-susceptible and moxalactam-resistant. CONCLUSIONS: Although all DD methods studied here performed well for detecting methicillin resistance in CoNS, moxalactam had a higher accuracy than cefoxitin to differentiate heteroresistant isolates from PBP2a-negative strains. Identification of isolates that should be submitted to a confirmatory test to conclude on methicillin resistance can be easily obtained by combining cefoxitin and moxalactam DD methods.

Unité(s) : Laboratoire de Microbiologie, Biostatistique

Age-related differences in the pharmacokinetics of stavudine in 272 children from birth to 16 years: a population analysis

JULLIEN V, RAIS A, URIEN S, DIMET J, DELAUGERRE C, BOUILLON-PICHAULT M, REY E, PONS G, BLANCHE S, TRELUYER JM

2007 - Br. J. Clin. Pharmacol. 64(1):105-109

AIMS: To develop a population pharmacokinetic model for stavudine in children and to investigate the consistency of the currently recommended dose based on adult target concentrations. METHODS: The pharmacokinetics of stavudine were investigated using a population approach. Individual estimates of CL/F were used to calculate the stavudine dose required to achieve the area under the concentration-time curve reported in adults given recommended doses. RESULTS: Stavudine pharmacokinetics were well described by a one-compartment model with zero-order absorption. Typical population estimates (% interindividual variability) of the apparent distribution volume (V/F) and plasma clearance (CL/F) were 40.9 l (32%) and 16.5 l h(-1) (38%), respectively. Stavudine V/F and CL/F were similarly related to age. Mean calculated doses (0.61 mg kg(-1) for children less than 2 weeks, 1.23 mg kg(-1) for children more than 2 weeks with bodyweight less than 30 kg, and 31.5 mg for children with a bodyweight between 30 and 60 kg) were in agreement with the current paediatric doses (0.5 mg kg(-1), 1 mg kg(-1), and 30 mg, respectively). CONCLUSIONS: Our findings support the current recommended paediatric dosage regimens for stavudine, as they result in the same exposure to the drug as in adults.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie, EA3620

HHV-6 and EBV DNA quantitation in lymph nodes of 86 patients with Hodgkin's lymphoma

LACROIX A, JACCARD A, ROUZIOUX C, PIGUET C, PETIT B, BORDESSOULE D, RANGER-ROGEZ S

2007 - J. Med. Virol. 79(9):1349-1536

Human herpesvirus (HHV-6) and Epstein-Barr virus (EBV), are two ubiquitous human herpesviruses which share many common features although they belong to different sub-families. In particular, both viruses are found in lymph nodes of patients suffering from Hodgkin's lymphoma. The aim of this study was to detect and to quantify independently HHV-6 and EBV by a real-time PCR in lymph nodes from 86 patients with Hodgkin's lymphoma. EBV quantitative method was compared with LMP-1 protein detection among the same samples. EBV genome was detected for 61.6% of the patients (53/86) and the highest prevalence of this virus was observed in Hodgkin's lymphoma with mixed-cellularity histopathological type (80%). In contrast to that, HHV-6 genome was detected for 79.1% of the patients (68/86) and was most observed in the nodular-sclerosis group (83.6%). Among the 68 HHV-6 positive samples, 63 belonged to the B subtype. A large number of biopsies (47.7%) were positive for both viruses whereas a little number (7%) was negative for both. EBV quantitation and LMP-1 immunohistochemistry were correlated statistically but this latter technique was less sensitive. Among the nodular-sclerosis patients, HHV-6-/EBV+ patients were significatively older than HHV-6+/EBV- patients. Patients infected dually had higher values of quantitation for each virus than those positive for one virus. Data of the clinical follow-up obtained by diagnosis and during the treatment of 83 patients, were correlated with the virological findings.

Unité(s) : Laboratoire de Microbiologie

Replication-competent HIV strains infect HIV controllers despite undetectable viremia (ANRS EP36 study)

LAMINE A, CAUMONT-SARCOS A, CHAIX ML, SAEZ-CIRION A, ROUZIOUX C, DELFRAISSY JF, PANCINO G, LAMBOTTE O

2007 - AIDS 21(8):1043-1045

The replicative potential of HIV-1 strains in a well-characterized group of eight HIV controllers was investigated. Replication-competent viruses were detected in CD4 T-cell co-culture supernatants from all HIV controllers. The phylogenetic analysis of C2V4 suggested viral evolution or co-infection or superinfection in two out of the four patients analysed. The vif and vpr genes were normal. Infection with HIV-1 variants with attenuated replicative capacity cannot be a general factor accounting for undetectable viraemia in HIV controllers.

Unité(s) : Laboratoire de Microbiologie

Multi-center trials need to use the same assay for hepatitis C virus (HCV) viral load determination

LAPERCHE S, BOUCHARDEAU F, THIBAULT V, POZZETTO B, VALLET S, ROSENBERG AR, ROQUE-AFONSO AM, GASSIN M, STOLL-KELLER F, TRIMOULET P, GAULT E, CHANZY B, MERCIER B, BRANGER M, PAWLOTSKY JM, HENQUELL C, LUNEL F, GAUDY-GRAFFIN C, ALAIN S, CHAIX ML, DUVERLIE G, IZOPET J, LEFRERE JJ

2007 - J. Clin. Microbiol. 45(11):3788-3790

This study involving twenty laboratories using currently available assays for HCV-RNA quantification demonstrates that differences in viral load values are not due to inter-laboratory variations but rather to the nature of the assay itself. This underlines the importance of using the same assay in multi-center studies or when monitoring antiviral therapy.

Unité(s) : Laboratoire de Microbiologie

Improved virological response to highly active antiretroviral therapy in HIV-1-infected patients carrying the CCR5 Delta32 deletion

LAURICHESSE J, PERSOZ A, THEODOROU I, ROUZIOUX C, DELFRAISSY J, MEYER L

2007 - HIV Med. 8(4):213-219

Background Patients heterozygous for the C-C chemokine receptor 5 (CCR5) Delta32 deletion spontaneously progress less rapidly to AIDS and death than do wild-type patients. We investigated whether the CCR5 Delta32 deletion has an impact on immunological, virological and clinical responses to highly active antiretroviral therapy (HAART) in HIV-1-infected patients. Patients and methods We included in the study 565 HIV-1-infected patients from the French HIV-1 infected cohort with documented date of serconversion (SEROCO)/haemophiliacs HIV-1 infected (HEMOCO) cohorts, who started HAART after 1996. We investigated virological responses to HAART at 6 months (defined as a plasma HIV-1 RNA measurement below the threshold of detection or a 2 log HIV-1 RNA decrease) and at 12 months (defined as a plasma HIV-1 RNA measurement below the threshold of detection) and clinical response to HAART by Kaplan-Meier survival curves, with AIDS and death as outcomes. Results The Delta32 heterozygous patients (n=83; 15%) had a better virological response to HAART than wild-type patients (73 vs 53% at 6 months, P=0.01; and 60 vs 44% at 12 months, P=0.01). This better virological response was still observed after adjustment for antiretroviral status (whether or not patients were naive to antiretroviral therapy), year of HAART initiation, number of new antiretroviral drugs and baseline viral load. There was no statistical difference between heterozygous patients and wild-type patients in terms of survival and AIDS-free survival. Conclusions CCR5 Delta32 heterozygous patients were more likely to have a virological response to HAART than wild-type patients at 6 and 12 months. However, this virological response did not produce better immunological and clinical responses. The long-term impact of the Delta32 deletion on survival in HIV-1-infected treated patients should be investigated in a meta-analysis.

Unité(s) : Laboratoire de Microbiologie

Diagnosis of viral respiratory infections

LERUEZ-VILLE M

2007 - Archives Pédiatrie 14(4):404-409

More than 80% of the cases of respiratory infections in children are of viral origin. Viral culture has been the reference method for the diagnosis of viral respiratory infections for years, but there is now a tendency to replace viral culture by molecular biology techniques, notably real-time PCR-based assay, because of its excellent sensitivity and good feasibility. Currently in most laboratories, however, diagnosis of viral respiratory infections is still done using techniques based on detection of viral antigens, especially immunofluorescence assays. Rapid diagnostic tests for use outside of laboratories are now available on the open market, and even if their sensitivity remains lower than that of other techniques, it is likely that they will become widely used, especially in doctors' offices, in the near future. New methods for the diagnosis of viral infections based on DNA microarray technologies are currently under investigation and appear to very promising.

Unité(s) : Laboratoire de Microbiologie, EA3620

Reversibility of cirrhosis in HIV/HBV coinfection

MALLET VO, DHALLUIN-VENIER V, VERKARRE V, CORREAS JM, CHAIX ML, VIARD JP, POL S

2007 - Antivir. Ther. 12(2):279-283

HIV infection worsens the course and the natural history of chronic hepatitis B (HBV) leading to rapid progression to cirrhosis and to end-stage liver disease. Highly active antiretroviral therapy (HAART) regimens including nucleoside and/or nucleotide analogues with activity against both HIV reverse transcriptase and hepatitis B virus polymerase have clearly improved the survival rates of HIV/HBV-coinfected patients. How HAART beneficially affects the natural course of chronic hepatitis B in coinfected patients is not known. We report a biopsy-proven case of reversal of HBV-related cirrhosis in a coinfected patient, paralleling long-term suppression of HBV replication with tenofovir disoproxil fumarate as part of a HAART. Pathological reversibility of cirrhosis was ascertained by normalization of biochemical (platelet count) and morphological (abdominal ultrasonography and gastrointestinal endoscopy) tests as well as non-invasive markers of fibrosis. In conclusion, a HAART regimen including tenofovir disoproxil fumarate in a HBV/HIV-coinfected cirrhotic patient might lead to sustained HBV viral suppression and result in cirrhosis reversal.

Unité(s) : Infectiologie, Laboratoire de Microbiologie, Radiologie Adulte, Anatomie Pathologique

Platelet Count Decline: An Early Prognostic Marker in Critically Ill Patients with Prolonged ICU Stays

MOREAU D, TIMSIT JF, VESIN A, GARROUSTE-ORGÉAS M, DE LASSENCE A, ZAHAR JR, ADRIE C, VINCENT F, COHEN Y, SCHLEMMER B, AZOULAY E

2007 - Chest 131(6):1735-1741

BACKGROUND Thrombocytopenia is common in intensive-care-unit (ICU) patients. The objective of this study was to evaluate possible links between declining platelet counts early in the ICU stay and survival. METHODS: All patients who were admitted for at least 5 days and had no thrombocytopenia at admission were included. A multivariable logistic regression model with hospital mortality as the outcome variable was built. RESULTS: We included 1077 patients. At admission, median platelet count was not significantly different in survivors (256 [interquartile range, 206-330].10(9)/L) and nonsurvivors (262 [211-351] .10(9)/L). Median SAPSII score at admission was worse in nonsurvivors (50 [37-63] versus 37 [27-48] in survivors, P<0.0001), as was the mean SOFA score on day 3 (6.3+/-3.24 versus 4+/-2.8, P<0.0001). Absolute platelet counts were lowest on day 4, but differed significantly between survivors and nonsurvivors only on day 7. Conversely, any percentage decline in platelet counts from 10% to 60% on day 4 was significantly associated with mortality. By multivariable analysis, a 30% decline in platelet count independently predicted death (OR 1.54, 95% CI [1.12-2.14], P=0.008), in addition to increasing or stable SOFA from admission to day 4, older age, male gender, admission for coma, worse SAPSII score at admission, transfer from another ward and co-morbidity. CONCLUSION: In patients who spend more than 5 days in the ICU and have normal platelet counts at admission, a decline in platelet counts provides prognostic information. This parameter deserves to be included in new scoring systems.

Unité(s) : Laboratoire de Microbiologie

Molecular Phylogenetics of Candida albicans

ODDS FC, BOUGNOUX ME, SHAW DJ, BAIN JM, DAVIDSON AD, DIOGO D, JACOBSEN MD, LECOMTE M, LI SY, TAVANTI A, MAIDEN MC, GOW NA, D'ENFERT C

2007 - Eukaryot. Cell 6(6):1041-1052

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.

Unité(s) : Laboratoire de Microbiologie

Comments on "Cytomegalovirus (CMV)-Encoded UL144 (Truncated Tumor Necrosis Factor Receptor) and Outcome of Congenital CMV Infection"

PICONE O, COSTA JM, CHAIX ML, VILLE Y, ROUZIOUX C, LERUEZ-VILLE M

2007 - J. Infect. Dis. 196(11):1719-1720

Unité(s) : Laboratoire de Microbiologie

Evaluation of a new mobile system for protecting immune-suppressed patients against airborne contamination

POIROT JL, GANGNEUX JP, FISCHER A, MALBERNARD M, CHALLIER S, LAUDINET N, BERGERON V

2007 - Amer. J. Infect. Control 35(7):460-466

BACKGROUND: Invasive aspergillosis is one of the most lethal airborne dangers for immune-suppressed subjects. Providing patient protection from such airborne threats requires costly and high-maintenance facilities. We herein evaluate a new self-contained mobile unit as an alternative for creating a patient protective environment. METHODS: Airborne contamination levels were monitored for different simulated scenarios and under actual clinical conditions. Functional tests were used to challenge the unit under adverse conditions, and a preliminary clinical study with patients and staff present was performed at 2 different French hospitals. RESULTS: Functional tests demonstrated that the unit can rapidly decontaminate air in the protected zone created by the unit and in the surrounding room. In addition, the protected zone is not sensitive to large disturbances that occur in the room. The clinical study included 4 patients with 150 accumulated days of testing. The protected zone created by the unit systematically provided an environment with undetectable airborne fungal levels (ie, <1 CFU/m(3)) regardless of the levels in the room or corridor (P < .01). CONCLUSIONS: These tests show that the unit can be used to create a mobile protective environment for immune-suppressed patients in a standard hospital setting.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie

Evaluation of Moxalactam with the BD Phoenix System for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci

PUPIN H, RENAUDIN H, JOIN-LAMBERT O, BEBEAR C, MEGRAUD F, LEHOURS P

2007 - J. Clin. Microbiol. 45(6):2005-2008

The performance of moxalactam with the BD Phoenix system for the detection of methicillin resistance in coagulase-negative staphylococci was evaluated by use of a collection of 186 strains. Moxalactam was a better drug as an indicator of methicillin resistance for mecA-positive strains than oxacillin and cefoxitin were. For strains other than Staphylococcus saprophyticus, a moxalactam MIC >16 mug/ml was indicative of methicillin resistance.

Unité(s) : Laboratoire de Microbiologie

Multidrug-resistant bacteria in hospitalized children: a 5-year multicenter study

RAYMOND J, NORDMANN P, DOIT C, VU THIEN H, GUIBERT M, FERRONI A, AUJARD Y

2007 - Pediatrics 119(4):e798-e803

OBJECTIVE: The objective of this study was to determine the incidence of multidrug-resistant bacteria in hospitalized children. METHODS: This multicenter study was conducted in 5 hospitals in the Paris area from 1999 to 2003. We recorded all isolations of multidrug-resistant bacteria from clinical samples that were obtained from hospitalized children. Strains that were isolated during systematic screening for carriers were excluded. RESULTS: The mean incidences were 0.9 per 1000 hospitalization-days for methicillin-resistant Staphylococcus aureus, 0.45 for extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, 0.32 for extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae, 0.40 for Enterobacter species with derepressed cephalosporinase, and 0.01 for vancomycin-resistant Enterococcus. The incidences per 1000 hospitalization-days of methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae, and Enterobacter species with derepressed cephalosporinase decreased significantly from 1999 to 2003, whereas the incidence of vancomycin-resistant Enterococcus remained very low. The proportion of resistant strains within the species did not vary significantly for methicillin-resistant Staphylococcus aureus (11% to 9.6%), extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae (1.1%), and vancomycin-resistant Enterococcus (0.03% to 0.023%). In contrast, the frequency of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae decreased from 31.6% to 7.4%, and that of Enterobacter species with derepressed cephalosporinase decreased from 38.8% to 18.5%. CONCLUSIONS: We report significant decreases in the incidence of methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, extended-spectrum beta-lactamase-producing Enterobacteriaceae other than Klebsiella pneumoniae, and Enterobacter species with derepressed cephalosporinase in hospitalized children during a 5-year period.

Unité(s) : Laboratoire de Microbiologie

Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children

ROSEY AL, ABACHIN E, QUESNES G, CADILHAC C, PEJIN Z, GLORION C, BERCHE P, FERRONI A

2007 - J. Microbiol. Meth. 68(1):88-93

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.

Unité(s) : Laboratoire de Microbiologie, Traumatologie et Orthopédie Pédiatriques

Impact of HIV-1 Genetic Diversity on Plasma HIV-1 RNA Quantification: Usefulness of the Agence Nationale de Recherches sur le SIDA Second-Generation Long Terminal Repeat-Based Real-Time Reverse Transcriptase Polymerase Chain Reaction Test

ROUET F, CHAIX ML, NERRIENET E, NGO-GIANG-HUONG N, PLANTIER JC, BURGARD M, PEETERS M, DAMOND F, EKOUEVI DK, MSELLATI P, FERRADINI L, RUKOBO S, MARECHAL V, SCHVACHSA N, WAKRIM L, RAFALIMANANA C, RAKOTOAMBININA B, VIARD JP, SEIGNEURIN JM, ROUZIOUX C

2007 - JAIDS 45(4):380-388

The high genetic diversity of HIV-1 has a major impact on the quantification of plasma HIV-1 RNA, representing an increasingly difficult challenge. A total of 898 plasma specimens positive for HIV-1 RNA by commercial assays (Amplicor v1.5; Roche Diagnostic Systems, Alameda, CA or Versant v3.0; Bayer Diagnostics, Emeryville, CA) were tested using the Agence Nationale de Recherches sur le SIDA second-generation (G2) real-time reverse transcriptase polymerase chain reaction (RT-PCR) test: 518 samples containing HIV-1 of known subtype, including 88 from 2 subtype panels and 430 harboring B (n = 266) and non-B (n = 164) group M HIV-1 subtypes from patients followed up in 2002 through 2005 at Necker Hospital (Paris, France), and 380 samples from 10 different countries (Argentina, Cambodia, Cameroon, Central African Republic, France, Ivory Coast, Madagascar, Morocco, Thailand, and Zimbabwe). HIV-1 RNA values obtained by G2 real-time PCR were highly correlated with those obtained by the Amplicor v1.5 for B and non-B subtypes (R = 0.892 and 0.892, respectively) and for samples from diverse countries (R = 0.867 and 0.893 for real-time PCR vs. Amplicor v1.5 and real-time PCR vs. Versant v3.0, respectively). Approximately 30% of specimens harboring non-B subtypes were underquantified by at least -0.51 log10 in Amplicor v1.5 versus 5% underquantified in G2 real-time PCR. Discrepant results were also obtained with subtype B samples (14% underquantified by Amplicor v1.5 vs. 7% by G2 real-time PCR). Similar percentages were observed when comparing results obtained with the G2 real-time PCR assay with those obtained using the Versant assay. Addressing HIV-1 diversity, continual monitoring of HIV-1 RNA assays, together with molecular epidemiology studies, is required to improve the accuracy of all HIV RNA assays.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

HIV-1 viral load testing cost in developing countries: what's new ?

ROUET F, ROUZIOUX C

2007 - Expert Rev. Mol. Diagn. 7(6):703-707

Unité(s) : Laboratoire de Microbiologie, EA3620

The measurement of HIV-1 viral load in resource-limited settings: how and where ?

ROUET F, ROUZIOUX C

2007 - Clin. Lab. 53(3-4):135-148

There is an urgent need for low-cost, simple, and accurate human immunodeficiency virus type 1 (HIV-1) viral load monitoring technologies in resource-limited settings, particularly at the time of the scaling-up of first and second-line highly active antiretroviral therapies. This review describes the main characteristics and advantages/ disadvantages of three alternative HIV-1 viral load methods currently evaluated and used in developing countries, i.e., the Ultra p24 antigen assay, the ExaVir Load reverse transcriptase activity test, and 'home-made' real-time PCR HIV-1 RNA techniques. This review discusses clinical results obtained with these three technologies in terms of correlation with commercial HIV-1 RNA assays, the impact of HIV-1 genetic diversity on quantification, as well as their usefulness for both the early diagnosis of pediatric HIV-1 infection and monitoring of highly active antiretroviral therapy efficiency. In addition, different strategies for HIV-1 viral load monitoring are discussed according to laboratory facilities in resource-constrained settings.

Unité(s) : Laboratoire de Microbiologie, EA3620

Examination of real-time PCR for HIV-1 RNA and DNA quantitation in patients infected with HIV-1 BF intersubtype recombinant variants

SCHVACHSA N, TURK G, BURGARD M, DILERNIA D, CAROBENE M, PIPPO M, GOMEZ-CARRILLO M, ROUZIOUX C, SALOMON H

2007 - J. Virol. Meth. 140(1-2):222-227

The impact of HIV-1 genetic diversity on the performance of laboratory testing is an issue that has to be monitored continuously. An "in-house" real-time PCR assay was developed by the Agence Nationale de Recherche sur le SIDA (ANRS) in France for viral load (VL) quantitation based on the amplification of the HIV-1 long terminal repeat (LTR) region. This technology has not been used in Argentina yet and considering the HIV-1 diversity in the country, a comparative analysis of this assay was undertaken versus the Versant HIV-1 RNA 3.0 Assay (b-DNA). The performance was assessed on 30 drug-naive HIV-1 infected patients who were characterized previously by phylogenetic analysis of the pol and vpu gene. The results showed that there is a significant linear correlation between values of transformed viral load logarithms measured by both, bDNA and real-time PCR assay and that this assay can be used to quantify viral load in samples from BF-infected patients with the same accuracy and reliability as for B subtype samples. The use of "in-house" real-time PCR to measure DNA in PBMCs correlated strongly with the HIV-1 RNA levels in all specimens.

Unité(s) : Laboratoire de Microbiologie

Wells' syndrome after primoinfection by parvovirus B19 in a child

TOULON A, BOURDON-LANOY E, HAMEL D, FRAITAG S, LERUEZ-VILLE M, DE PROST Y, HADJ-RABIA S

2007 - J. Amer. Acad. Dermatol. 56(2 Suppl.):S50-S51

Unité(s) : Dermatologie, Anatomie Pathologique, Laboratoire de Microbiologie

Evaluation of CMV DNA quantification in dried blood spots: retrospective study of CMV congenital infection

VAULOUP-FELLOUS C, DUCROUX A, COULOIGNER V, MARLIN S, PICONE O, GALIMAND J, LOUNDON N, DENOYELLE F, GRANGEOT-KEROS L, LERUEZ-VILLE M

2007 - J. Clin. Microbiol. 45(11):3804-3806

We compared two DNA extraction protocols from dried blood spots (DBS) for CMV DNA detection and quantification by real time PCR. Both extraction methods were reliable for retrospective diagnosis of CMV congenital infection. Quantification of CMV DNA was valuable after normalization of viral loads with albumin gene PCR amplification results.

Unité(s) : ORL & Chirurgie Cervico-Faciale, Laboratoire de Microbiologie

Long-term nonprogression of HIV infection in children: evaluation of the ANRS prospective French Pediatric Cohort

WARSZAWSKI J, LECHENADEC J, FAYE A, DOLLFUS C, FIRTION G, MEYER L, DOUARD D, MONPOUX F, TRICOIRE J, BENMEBAREK Y, ROUZIOUX C, BLANCHE S

2007 - Clin. Infect. Dis. 45(6):785-794

BACKGROUND: Some children who are infected with human immunodeficiency virus type 1 (HIV-1) during the perinatal period remain asymptomatic for very long periods in the absence of antiretroviral treatment, as is the case for some adults. Our objective was to estimate the proportion of children who developed neither symptoms nor major immunological perturbations to the age of > or = 10 years in a prospective cohort of infected children who had been observed since birth. METHODS: The ongoing prospective French Pediatric Cohort includes 568 HIV-1-infected children. Here, we report the follow-up data for all 348 HIV-1-infected children who were born before 1 January 1994. Children with long-term nonprogression of infection (LTNPs) were defined as HIV-1-infected children who had been observed for at least 10 years, never received antiretroviral treatment other than zidovudine monotherapy, never developed symptoms of Centers for Disease Control and Prevention clinical category C or B, and had a CD4+ cell percentage of < 25% no more than once during follow-up. Other definitions were compared. RESULTS: The Kaplan-Meier estimate of long-term nonprogression was 2.4% (95% confidence interval, 1.1%-4.6%) at 10 years of age, and 7 children were classified as LTNPs. The Kaplan-Meier estimates decreased slightly with age, to 1.8% at 12 years of age and 1.4% at 14 years of age. Plasma HIV-1 replication rates were low (< 1000 copies RNA/mL) for 2 of the 7 LTNPs at the age of 10 years (0.6% of the total denominator). None of the routinely measured maternal or perinatal markers were significantly linked to long-term nonprogression, with the exception of the mother's Centers for Disease Control and Prevention clinical category at the time of delivery. CONCLUSIONS: Approximately 2% of children who were infected during the perinatal period displayed no immunological or clinical progression by the age of 10 years. This figure is close to that reported for adults in studies that have used similar definitions.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Laboratoire de Microbiologie, EA3620

Updating on nosocomial infections in France

ZAHAR JR

2007 - M S-Méd. Sci. 23(6-7):644-645

Unité(s) : Laboratoire de Microbiologie

Is it time to reconsider initial antibiotic treatment strategies for severe urinary tract infections in Europe ?

ZAHAR JR, LECUIT M, CARBONNELLE E, RIBADEAU-DUMAS F, NASSIF X, LORTHOLARY O

2007 - Clin. Microbiol. Infection 13(3):219-221

Until recently, most reported cases of bacteraemia caused by multidrug-resistant strains of Enterobacteriacae producing an extended-spectrum beta-lactamase (ESBL) in Europe have been nosocomial in origin. However, increasing numbers of reports of community-acquired bacteraemia and urinary tract infection caused by ESBL-producing microorganisms suggest that the geographical origin of patients should be taken into account as a risk-factor for possible ESBL production. Early identification of patients at high-risk of infection with ESBL-producing microorganisms, based on their geographical origin and travel history, should help to optimise initial antibiotic treatment strategies for severe urinary tract infections in Europe.

Unité(s) : Infectiologie, Laboratoire de Microbiologie

Stable recovery from HCV in HIV-HCV co-infection under antiretroviral therapy

ZEITOUN JD, MALLET V, CHAIX ML, VIARD JP, BLANCHE S, POL S

2007 - J. Clin. Virol. 40(1):71-73

Co-infection by the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) is common. HIV co-infection worsens the course of HCV infection, particularly in patients with low CD4-cell count, enhancing the need to cancel HCV replication in HIV-infected patients. We report a case of spontaneous recovery of chronic HCV-infection in a HCV-HIV co-infected hemophiliac with the introduction of combined antiretroviral therapy and immune restoration. Retrospective analysis of frozen blood samples showed that a decrease in HCV-RNA in parallel with the increase of CD4 and CD8-cell counts, accompanied by a peak of transaminases. The respective effect of immune restoration and of high doses of ritonavir are discussed.

Unité(s) : Immuno-Hématologie-Rhumatologie Pédiatriques, Infectiologie, Laboratoire de Microbiologie

Impact of highly active antiretroviral therapy on the maturation of anti-HIV-1 antibodies during primary HIV-1 infection

ADALID-PERALTA L, GRANGEOT-KEROS L, RUDENT A, NGO-GIANG-HUONG N, KRZYSIEK R, GOUJARD C, DEVEAU C, LE GALL M, MEYER L, EMILIE D, ROUZIOUX C

2006 - HIV Med. 7(8):514-519

Objectives To study the impact of highly active antiretroviral therapy (HAART) on isotype switching and avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with primary HIV-1 infection (PHI). Methods We studied the emergence and the evolution of anti-HIV IgG antibodies by quantitative immunoblotting to analyse IgG subclasses and IgG avidity. Serum samples were obtained from 16 PHI patients from the French PRIMO Cohort Study at various points in the first year of infection: eight patients received no treatment (group I), and eight patients received efficient HAART (group II) during the study period. Results Early initiation of HAART in PHI patients partially prevented an increase in anti-HIV-1 IgG levels. Within IgG subclasses, the amount of anti-HIV-1 IgG1 gradually increased with time in both groups, although levels remained lower in treated patients. The anti-p24 IgG2 level was always lower in group II. We observed a decrease in anti-p24 IgG3 over time in both groups. Treatment did not affect the maturation of HIV-1 IgG avidity, which increased in both groups until month 3 and then remained high until the end of the 12-month follow-up period. Conclusions HAART in PHI partially prevents the emergence of HIV-1 IgG antibodies, but does not affect the quality of these antibodies, as reflected in their isotype and avidity.

Unité(s) : Laboratoire de Microbiologie

Quantitation of HIV-1 RNA in breast milk by real time PCR

BECQUART P, FOULONGNE V, WILLUMSEN J, ROUZIOUX C, SEGONDY M, VAN DE PERRE P

2006 - J. Virol. Meth. 133(1):109-111

HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

Unité(s) : Laboratoire de Microbiologie, EA3620

Scientific progress and new biological weapons

BERCHE P

2006 - M S-Méd. Sci. 22(2):206-211

The biological weapons are different from conventional weapons, because living germs hold an extraordinary and predictable potential for multiplication, propagation and genetic variation during their dissemination in a susceptible population. Only natural pathogens (1rst generation weapons) have been used in the past (smallpox virus, plague, anthrax, toxins...). However, new threats are emerging, due to the rapid progress of scientific knowledge and its exponential worldwide diffusion. It is possible to synthesize microorganisms from in silico sequences widely diffused on Internet (poliovirus, influenza...), thus resulting in the accessibility of very dangerous virus confined today in high-security laboratories (virus Ebola...). It is possible also to << improve >> pathogens by genetic manipulations, becoming more resistant or virulent (2nd generation weapons). Finally, one can now create de novo new pathogens by molecular breeding (DNA shuffling), potentially highly dangerous for naive populations (3rd generation weapons). Making biological weapons does not require too much technological resources and appears accessible to terrorists, due to low cost and easy use. Although the destructive consequences are difficult to predict, the psychological and social damages should be considerable, because of the highly emotional burden in the population associated to the transgression by man of a taboo of life. double dagger.

Unité(s) : Laboratoire de Microbiologie

Multilocus sequence typing reveals intrafamilial transmission and microevolutions of Candida albicans isolates from the human digestive tract

BOUGNOUX ME, DIOGO D, FRANCOIS N, SENDID B, VEIRMEIRE S, COLOMBEL JF, BOUCHIER C, VAN KRUININGEN H, D'ENFERT C, POULAIN D

2006 - J. Clin. Microbiol. 44(5):1810-1820

Candida albicans is a human commensal that is also responsible for superficial and systemic infections. Little is known about the carriage of C. albicans in the digestive tract and the genome dynamics that occur during commensalisms of this diploid species. The aim of this study was to evaluate the prevalence, diversity, and genetic relationships among C. albicans isolates recovered during natural colonization of the digestive tract of humans, with emphasis on Crohn's disease patients who produce anti-yeast antibodies and may have altered Candida sp. carriage. Candida sp. isolates were recovered from 234 subjects within 25 families with multiple cases of Crohn's disease and 10 control families, sampled at the oral and fecal sites. Prevalences of Candida sp. and C. albicans carriage were 53.4% and 46.5%, respectively, indicating frequent commensal carriage. No differences in prevalence of carriage could be observed between Crohn's disease patients and healthy subjects. Multilocus sequence typing (MLST) of C. albicans isolates revealed frequent colonization of a subject or several members of the same family by genetically indistinguishable or genetically close isolates. These latter isolates differed by loss-of-heterozygosity events at one or several of the MLST loci. These loss-of-heterozygosity events could be due to either chromosome loss followed by duplication or large mitotic recombination events between complementary chromosomes. This study was the first to jointly assess commensal carriage of C. albicans, intrafamilial transmission, and microevolution. The high frequency of each of these events suggests that the digestive tract provides an important and natural niche for microevolutions of diploid C. albicans through the loss of heterozygosity.

Unité(s) : Laboratoire de Microbiologie

Antibodies to conserved epitopes of the HIV-1 envelope in sera from long-term non-progressors: prevalence and association with neutralizing activity

BRAIBANT M, BRUNET S, COSTAGLIOLA D, ROUZIOUX C, AGUT H, KATINGER H, AUTRAN B, BARIN F

2006 - AIDS 20(15):1923-1930

OBJECTIVE:: Previous studies have shown that broadly neutralizing antibodies (NAb) are more frequent in long-term non-progressors (LTNP) than in other HIV-1 infected patients, but nothing is known about the envelope regions targeted by these broadly NAb. We investigated whether the breadth of neutralizing activity of sera was associated with the presence of specific antibodies (2F5- and/or 4E10-like, b12-like or 2G12-like antibodies) directed against conserved epitopes known to be involved in broad neutralization. METHODS:: We assessed the ability of sera from 67 LTNP of the French ANRS cohort (ANRS CO15) to neutralize four heterologous primary isolates of four various clades. Competitive and non-competitive ELISA were developed for the specific comparison of levels of antibodies against these specific epitopes in neutralizing and non-neutralizing sera from LTNP. RESULTS:: We found that higher 2G12-like antibody levels were significantly associated with the broadest neutralizing activity in sera from LTNP. Levels of 2G12-like antibodies were higher in the sera that neutralized the four isolates than in the others, with a median of 5.7 mug/ml [int