Pandemic influenza A(H1N1) 2009: molecular characterisation and duration of viral shedding in intensive care patients in Bordeaux, south-west France, May 2009 to January 2010.

From May 2009 to January 2010, the Virology Laboratory at the University Hospital of Bordeaux received more than 4,000 nasopharyngeal samples from the Aquitaine region (south-west France) for the diagnosis of pandemic influenza A(H1N1)2009. Eighty-three infected patients deteriorated and were admitted to intensive care units. Our study focused on 24 of these patients. Positivity for influenza A(H1N1)2009 was monitored by realtime PCR and duration of viral shedding was determined. The first available sample of each patient was analysed for bacterial, fungal and viral co-infection. We observed six bacterial (or bacterial/fungal) co-infections and one viral co-infection with respiratory syncytial virus. The samples were analysed for the presence of the neuraminidase H275Y (N1 numbering) mutation, which confers resistance to oseltamivir, by realtime PCR of the neuraminidase gene. No H275Y mutation was observed in any of the viral strains screened in this study. In parallel, a fragment of the haemagglutinin gene encoding amino acid residues 173 to 362 was sequenced to detect mutations that had been reported to increase the severity of the disease. Two patients were infected by strains bearing the D222G (H3 numbering) mutation. The viral shedding of A(H1N1)2009 in this study ranged from four to 28 days with a median of 11 days.

[First cases of secondary transmission of a novel swine-origin influenza A (H1N1) virus in France]. / Premiers cas de transmission secondaire en France du nouveau virus grippal d'origine porcine A(H1N1)v.

On April 2009, a new swine-origin A(H1N1) influenza virus, A(H1N1)v, was identified in the United States. Today (June 12, 2009), more than 29,000 cases have been reported in the world, and 73 in France. This is the first report of secondary transmission in France. The three patients presented with common influenza signs including cough, fever, and sore throat. The incubation period could last from two to four days; it should be kept in mind that the first international data mentioned one to seven days. The buildup and maintenance of an infectious focus involve secondary transmissions.

Quantitation of HIV-1 RNA in dried blood and plasma spots.

Dried blood spots (DBSs) and dried plasma spots (DPSs) are an attractive method for serological and molecular diagnosis of HIV infection. Recently, Youngpairoj et al. [Youngpairoj, A.S., Masciotra, S., Garrido, C., Zahonero, N., de Mendoza, C., Garcia-Lerma, J.G., 2008. HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C. J. Antimicrob. Chemother. 61, 1217-1220] showed that HIV-1 can be genotyped efficiently from DBS specimens stored at 4 degrees C for 1 year. The viral load obtained from DBS and DPS samples was compared with that obtained from plasma samples. A total of 86 samples was prepared from people infected with HIV subtype B or non-B and spotted on 903 filter papers stored with desiccant at 4 degrees C. RNA was extracted using the QIAamp Viral RNA mini kit (Qiagen, Courtaboeuf, France). RNA from DBS or DPS samples was quantified in accordance with the Agence Nationale de Recherche sur le SIDA (ANRS, AC11, Paris, France) assay for HIV-1 quantitation. When the mean viral load of plasma samples and DPS samples or plasma samples and DBS samples were compared, there were no significant differences. The overall data showed that although the sensitivity threshold of the assays was different, there was a correlation between the three specimen types and that DBS and DPS samples can be routinely used for viral load quantification particularly in resource-limited settings.

PCR-detected hepatitis C virus RNA associated with human zona-intact oocytes collected from infected women for ART.

BACKGROUND: The aim of this study was to investigate the susceptibility of human oocytes from hepatitis C virus (HCV) RNA-positive women to HCV contamination during assisted reproductive technology (ART). METHODS: A reverse transcriptase-PCR assay was used to test for the presence of HCV RNA associated with 24 unfertilized oocytes 48 h after follicular fluid aspiration in 10 IVF attempts (seven conventional IVF and three ICSI). Negative and positive controls (10 unfertilized oocytes from HCV-negative women and 20 unfertilized oocytes artificially contaminated with HCV RNA-positive plasma; HCV RNA was also quantified in plasma and follicular fluid) were included. RESULTS: HCV RNA was associated with 17/24 (70.8%) oocytes (6/7 after ICSI and 11/17 after conventional IVF) and was found in 19/20 (95%) follicular fluid samples. A weak correlation was found between plasma and follicular fluid HCV RNA loads (r = 0.73, P < 0.001). CONCLUSIONS: HCV associated with unfertilized oocytes surrounded by their intact zona pellucida from anti-HCV antibody-positive and viraemic women undergoing ART raises questions concerning the safe management of medically assisted procreation for these women and good practice of oocyte/embryo cryopreservation and donation.

Mechanism of virologic failure after substitution of a protease inhibitor by nevirapine in patients with suppressed plasma HIV-1 RNA.

A prospective study was set up to evaluate the emergence of HIV-1 resistance after a switch from an effective protease inhibitor (PI)-containing regimen to a multitherapy regimen including nevirapine (NVP). After 6 months with an undetectable viral load under a PI-containing regimen, the patients were switched to NVP with conservation of the associated nucleoside reverse transcriptase inhibitors (NRTIs). Patients were followed-up at 1 month and then every 3 months after switching therapy. Nucleotide sequence analysis of the pol gene was performed at the first points of virologic failure. Thirty-four patients were included. The NRTI-naive group (22 patients) had begun antiretroviral therapy with a PI-containing regimen, whereas 12 patients (experienced group) had been previously treated by nucleoside mono-and/or dual therapy. After a median follow-up of 40 weeks, no patient of the naive group, versus 41% of the experienced group, developed a virologic failure after the change toward NVP ( p =.003). The virologic failures were associated with the appearance of NNRTI-resistant mutations. All rebound mutants also presented NRTI-resistance mutations. These results are consistent with a higher risk of virologic failure after a switch to an NNRTI in patients with prior suboptimal treatment and suggest the hypothesis that archived resistant viruses may facilitate the emergence of NNRTI resistance.

Sequence analysis of env C2/V3, gag p17/p24, and pol protease regions of 25 HIV type 1 isolates from Ho Chi Minh City, Vietnam.

Env C2/V3, gag p17/p24, pol protease, and RT regions of HIV-1 isolates recently obtained from 25 HIV-1 seropositive individuals from Ho Chi Minh City (Vietnam) were studied, and genes subtypes were determined by DNA sequence analyses. Twenty-three isolates out of 25 were identified as belonging to subtype E, now recognized as circulating recombinant form 1 (CRF01_AE). The motif at the top of the V3 loop (generally GPGQ) was then preceded by an isoleucine or a methionine (M) residue; the M residue might be a local signature of Vietnamese E isolates compared to Thai E viruses. Two isolates (8%) were shown to be intersubtype recombinants: one E/B and one CRF02_AG(IBNG)/D. The polymorphism of pol protease was considered only for CRF01_AE isolates and is clearly different from that recorded for B viruses with substitutions at positions 13, 35, 36, 41, 69, and 89.

Pharmacokinetics and resistance mutations affect virologic response to ritonavir/saquinavir-containing regimens.

The authors assessed the impact of protease and reverse transcription (RT) mutations and individual pharmacokinetic parameters on virologic response to a four-drug regimen including ritonavir/saquinavir. Treatment was given at the start of the study (M0) to 22 HIV-1 protease inhibitor-naive or pretreated patients. Protease and RT genes were sequenced at M0, at the time of virologic failure, or at the end of the follow-up. Plasma ritonavir and saquinavir peak C(max), C(min), and area under the curve (AUC) were determined based on samples taken 0, 1, 2, 3, 4, 6, 8, and 12 hours after administration. HIV-1 RNA decreased to less than 50 copies/mL in 11 patients (group 1). At M0, five of them had no RT mutation and 10 had three or fewer secondary protease mutations with no new mutation during follow-up. Ritonavir and saquinavir pharmacokinetics showed wide interindividual variability. Treatment failed in 11 patients (group 2): 9 had three to eight protease mutations and a mean of 5.8 RT mutations at M0, with emergence of new mutations during follow-up. Pharmacokinetics was similar to those of group 1. The other two patients with virologic failure showed no baseline primary mutation but were the only patients with insufficient saquinavir and ritonavir AUC. The authors showed the complementarity between drug-resistance genotype and individual pharmacokinetics and the potential utility of AUC and Cmax to manage treatment.

Hepatitis C virus genotyping based on 5' noncoding sequence analysis (Trugene).

Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5'NC genotyping kit, based on sequence analysis of the 5' noncoding region (5' NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5' NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.

Qualitative and quantitative molecular detection of enteroviruses in water from bathing areas and from a sewage treatment plant.

Pathogenic enteric viruses can be introduced into the environment as a result of human activities. Enteroviruses are regularly detected in environmental waters or shellfish and can provoke potentially serious diseases. Some authors believe that enteroviruses could represent an interesting indicator of viral contamination in the environment. Since molecular approaches seem to be promising for the detection of these viruses, we developed a simple qualitative RT-PCR procedure for enteroviruses, together with a quantitative RT-PCR assay using RNA internal standard. After one-tube-RT-PCR, this standard and wild enterovirus RNA were detected by differential hybridization with specific probes and a fluorimetric reaction. The quantification of enteroviruses, conducted in a sewage treatment plant, showed a decreasing number of genomic copies from the entrance to the exit (from 3.8 x 10(5) to 5.4 x 10(4) RNA copies/mL) but indicated the presence of enterovirus RNA in the neighboring river (2.2 x 10(3) RNA copies/mL). In bathing areas, enterovirus RNA was detected in 16 out of 226 samples, with copies numbers ranging from 3.7 x 10(2) RNA copies/mL to 7 x 10(4) RNA copies/mL.

PCR detection of human enteric viruses in bathing areas, waste waters and human stools in Southwestern France.

Detection of human pathogenic viruses by molecular techniques might be suitable for identifying viral pollution in environmental waters and for improving diagnosis in patients. Environmental samples were taken from bathing areas and sewage treatment plants in southwestern France. Small volume samples (50 microL) were tested. Five groups of enteric pathogenic viruses were studied: enteroviruses, Norwalk-like viruses (NLVs), hepatitis A virus, rotaviruses and adenoviruses. Moreover, human samples were tested for NLV. After extraction of viral nucleic acids (Boom's procedure), a nested polymerase chain reaction was conducted before hybridization. Five bathing waters out of 26 were positive for one viral group, without systematic association with bacterial contamination. Eight sewage plant samples out of 13 were positive for at least one viral group. Seven patients out of 45 were NLV-positive. Molecular techniques allow efficient screening of viral contamination in environmental waters and the study of NLV molecular epidemiology.

Hepatitis B exacerbation with a precore mutant virus following withdrawal of lamivudine in a human immunodeficiency virus-infected patient.

Chronic active hepatitis B exacerbations have been reported following development of resistance to or withdrawal of lamivudine in HIV-infected patients. A 38-year-old woman with HIV and chronic HBV infections was hospitalized because of acute hepatitis. The occurrence of cytolysis with replication of HBV 2 months after withdrawing lamivudine suggests that our patient experienced a severe reactivation of HBV infection due to the modification of her treatment. Sequencing of the HBV precore region showed the strain to be a mutant. We conclude that lamivudine should not be stopped in HIV- and HBV-infected patients, but could be continued at the dose of 100mg/day as used in isolated HBV infection.

Asymmetric periflexural exanthem of childhood: microbiologic case-control study.

Our objective was to study possible etiologic factors of asymmetric periflexural exanthem of childhood (APEC) among a large panel of microbiologic agents not yet investigated. To do so, we designed a prospective case-control study using throat, stool, blood, and skin samples, and enlisted 37 children with APEC and 37 age-matched controls without eruption seen consecutively from February 1995 to April 1996 from a mixed referral center and community-based population. No interventions were done. Used as the main outcome measure was the differences in the two groups for microbiologic investigations. No significant statistical differences between cases and controls for virus and bacteria investigated were found. No microorganism was identified as a possible etiologic agent in any of the APEC patients. APEC is not a nonspecific cutaneous eruptive pattern to several common microbiologic agents. More sophisticated molecular approaches are needed to address its etiology.

A hospital outbreak of gastroenteritis possibly related to the contamination of tap water by a small round structured virus.

Small round structured viruses (SRSVs) are a major cause of gastroenteritis in institutions and sensitive new molecular techniques allow rapid diagnosis and the establishment of control measures. In January 1999, a 10 day-long outbreak of gastroenteritis in a re-education ward, was reported by a hospital hygiene department. A potential common source of contamination was tap water. The stools of six patients with gastroenteritis and seven tap water samples from the hospital ward, were tested for SRSV by reverse transcription and polymerase chain reaction (RT-PCR): three stools and four water samples, all bacteriologically negative, were SRSV-positive. Nucleotide sequencing of a fragment of the SRSV polymerase gene showed that the sequences of the positive samples (two patients and four water samples) were identical (genogroup II). We cannot exclude interhuman transmission of SRSV together with viral soiling of some taps in the ward, but this hospital infection was more likely due to the transient contamination of the ward supply of drinking water with a SRSV strain.

Clinical and biological characteristics of human immunodeficiency virus-infected and uninfected intravascular drug users in Ho Chi Minh City, Vietnam.

To define the medical characteristics of intravascular drug users in Ho Chi Minh City, Vietnam, we examined 280 men, of whom 235 were infected with human immunodeficiency virus (HIV), being treated in a rehabilitation center. The patients used mainly opium, often in shooting galleries (50%). The prevalence of oral candidiasis (58%) and zoster infection (20%) was high in HIV-seropositive patients, whereas oral hairy leukoplasia and Kaposi's sarcoma were absent. The prevalence of acquired immunodeficiency syndrome was 24%. More than 80% of the patients had infections with hepatitis C virus, hepatitis B virus, cytomegalovirus, or human T cell lymphotropic virus type-1. The CD4+ cell counts correlated well with viral load. Only HIV-1 subtype E was detected in the 30 patients tested. A cohort study of HIV-infected subjects in this population seems feasible, and would permit introduction of anti-retroviral therapy The large number of HIV-seronegative subjects sharing the same at-risk practices as the HIV-infected subjects raises the possibility of natural protection in this population.

Human polyomavirus JC latency and reactivation status in blood of HIV-1-positive immunocompromised patients with and without progressive multifocal leukoencephalopathy.

BACKGROUND: Human polyomavirus JC (JCV) induces human progressive multifocal leukoencephalopathy (PML) in patients with AIDS. Peripheral blood mononuclear cells (PBMC) of HIV-1-positive immunocompetent and immunocompromised patients can harbour JCV genome, but their precise role in JCV latency or reactivation status before the onset of PML remains hypothetical. OBJECTIVES: To assess JCV latency or reactivation status in PBMC of HIV-1-positive immunocompromised patients without PML. DESIGN: A group of 82 HIV-1-positive immunocompromised patients who did not have PML were compared with 10 patients with AIDS and PML and with 69 HIV-1-positive immunocompetent patients without PML. METHODS: DNA and total RNA were extracted from PBMC. The presence of JCV DNA was demonstrated by a semi-nested polymerase chain reaction (PCR). By using primer pairs specific for an early gene,T, and a late gene, VP1, the expression of both early and late gene mRNA in PBMC could be identified using reverse transcriptase (RT) PCR. RESULTS: JCV DNA was detected by PCR in 17.4% of 69 HIV-1-positive immunocompetent patients, in 23.2% of 82 HIV-1-positive immunocompromised patients, and in 60% of 10 patients with AIDS and PML. No correlation could be drawn between the detection of JCV DNA in the PBMC and the clinical or biological status of the HIV-1-positive patients. By using RT-PCR procedures, no expression of JCV early and late mRNA in PBMC was found in any patients. CONCLUSIONS: JCV DNA is detectable in the PBMC of 20.5% of 151 HIV-1-infected patients independently of the CDC (Centers for Disease Control and Prevention) stages of the infection. Moreover, our results suggest that active replication of JCV in PBMC appears to be absent or at least a very rare event in HIV-1-positive immunocompromised patients with and without PML.

Effect of nucleoside analogs and non-nucleoside inhibitors of HIV-1 reverse transcriptase on cell-free virions.

Reverse transcription takes place in the cytoplasm of infected cells, although it has been demonstrated that retroviruses can also initiate reverse transcription prior to infection of target cells. In addition to partial reverse transcripts, full-length proviral molecules have been detected in the plasma and seminal fluid of HIV-1 seropositive patients. Intravirion endogenous reverse transcription appears to be directly correlated with an increased level of infectivity. Therefore, the ability of an inhibitor to reach and inhibit the replication complex in the core of the free-virion may constitute an important part of its capacity to suppress viral infection. In this work we tested the ability of some reverse transcriptase inhibitors to decrease viral infectivity in pretreated highly purified virions. Our results showed that Curie pyridinone [Dollé et al. (1995), J Med Chem 38: 4,679-4,686], a non nucleoside RT inhibitor, strongly inhibited the infectivity of extracellular HIV-1 particles. Other non nucleoside inhibitors (TIBO R82913, HEPT, nevirapine) tested in these conditions were unable to do so. Our data indicate that the effect of Curie pyridinone on intact virions may be related to its capacity to tightly bind the target RT. This approach may lead to the design and synthesis of new drugs able to interact with the retroviral enzyme inside the viral core.

Zidovudine resensitization and dual HIV-1 resistance to zidovudine and lamivudine in the delta lamivudine roll-over study.

OBJECTIVE: To study zidovudine resensitization and dual resistance to zidovudine/lamivudine in HIV-1 isolates from nucleoside reverse transcriptase (RT) inhibitor-experienced patients during selective pressure exerted by zidovudine/lamivudine combination therapy. DESIGN AND METHODS: HIV-1 isolates from 29 patients receiving zidovudine/lamivudine combination therapy in the Delta roll-over study were analysed at entry and during a 1 year follow-up period for phenotypic susceptibility to zidovudine and lamivudine in the ANRS PBMC assay. The RT gene from codon 20 to 230 and at codon 333 was analysed by nucleotide sequencing of the corresponding isolates. RESULTS: HIV-1 isolates from 23 of the 29 patients were phenotypically resistant to zidovudine at baseline; 61% of these patients showed significant zidovudine resensitization during follow-up. The zidovudine IC50 value correlated positively with log10 plasma HIV-1 RNA (P = 0.02) and negatively with the CD4 cell count (P = 0.004). Zidovudine resensitization (related to acquisition of the M184V mutation) was transient, with evolution towards dual resistance to zidovudine and lamivudine in 20 of the 29 patients. The phenotype of certain dually resistant isolates coincided with the emergence of multiple mutations in the 5' part of the RT gene. CONCLUSIONS: M184V-mediated zidovudine resensitization of HIV-1 is transient in most patients who are given zidovudine/lamivudine combination therapy when zidovudine resistance has already emerged. The subsequent evolution towards dual phenotypic resistance to zidovudine/lamivudine corresponds to complex genotypic profiles.