[A case of fatal neonatal Bacillus cereus meningitis]. / Méningite néonatale à Bacillus cereus, à propos d'un cas.

A fatal case of meningoencephalitis caused by Bacillus cereus, an uncommon but potential pathogen, resistant to most beta-lactam antibiotics, is described in a 28-day old premature neonate. Difficulties for clinical diagnosis and treatment are discussed. A review of the literature (26 published cases) is given. Early diagnosis of neonatal B. cereus infection is crucial as it leads to a standard treatment including vancomycin.

Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: The GEMHEP GenoCII Study.

This cross-sectional study aimed to investigate, during a short period between 2000 and 2001, in a large population of patients with chronic hepatitis C, the epidemiological characteristics of hepatitis C virus (HCV) genotypes in France. Data from 26 referral centres, corresponding to 1769 patients with chronic hepatitis C were collected consecutively during a 6-month period. HCV genotyping in the 5'-non-coding region (NCR) was performed in each center using the line probe assay (LiPA, in 63% of cases), sequencing (25%) or primer-specific polymerase chain reaction (PCR) (12%). HCV genotypes 1a, 1b, 2, 3, 4, 5, non-subtyped 1 and mixed infection were found in 18, 27, 9, 21, 9, 3, 11 and 1% of our population, respectively. HCV genotype distribution was associated with gender, age, source and duration of infection, alanine aminotransferase (ALT) levels, cirrhosis, alcohol consumption, hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection. In multivariate analysis, only the source of infection was the independent factor significantly associated with genotype (P = 0.0001). In conclusion, this study shows a changing pattern of HCV genotypes in France, with i.v. drug abuse as the major risk factor, an increase of genotype 4, and to a lesser extent 1a and 5, and a decrease of genotypes 1b and 2. The modification of the HCV genotype pattern in France in the next 10 years may require new therapeutic strategies, and further survey studies.

Prevalence of resistance mutations in antiretroviral-naive chronically HIV-infected patients in 1998: a French nationwide study.

OBJECTIVE: To estimate the prevalence of resistance-conferring mutations to antiretroviral drugs in previously untreated patients with chronic HIV-1 infection as a basis for French recommendations on viral genotyping before antiretroviral treatment initiation. DESIGN: Resistance mutations were sought in samples from 404 patients seen in 23 specialized centres throughout metropolitan France in 1998. METHODS: The protease and reverse transcriptase (RT) genes of plasma virions were sequenced. Primary and secondary protease and RT gene mutations were identified from the International AIDS Society resistance testing - USA panel. RESULTS: The prevalence of patients with primary and secondary mutations were 3.7% (95% CI 1.7-5.7) and 50.3% (95% CI 45.0-55.6), respectively. The prevalence of patients with mutations associated with resistance to nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors was 3.3% (95% CI 1.5-5.1) and 0.8% (95% CI 0.0-1.7), respectively. The prevalence of patients with NRTI primary mutations differed according to whether seropositivity had been diagnosed more or less than one year previously (0.2 versus 2.2% P = 0.023). Primary mutations associated with protease inhibitor resistance occurred at a prevalence of 1.9% (95% CI 0.5-3.4) with no difference according to the duration of known seropositivity. CONCLUSION: In France, in 1998, the prevalence of patients with primary mutations associated with resistance to antiretroviral drugs was low. Genotyping before the initiation of therapy was not recommended in chronically HIV-1-infected naive patients. A national sentinel survey of resistance in this clinical setting is performed regularly to update the recommendations for resistance testing.

Genotypic and phenotypic resistance patterns of human immunodeficiency virus type 1 variants with insertions or deletions in the reverse transcriptase (RT): multicenter study of patients treated with RT inhibitors.

Genomic rearrangements in the 5' part of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been involved in multidrug resistance to nucleoside RT inhibitors (NRTI). We carried out a retrospective, multicenter study to investigate the prevalence, variability, and phenotypic consequences of such rearrangements. Data concerning the HIV-1 RT genotype and the biological and clinical characteristics of NRTI-treated patients were collected from 10 virology laboratories. Sensitivities of the different HIV-1 variants to RT inhibitors were analyzed in a single-cycle recombinant virus assay. Fifty-two of 2,152 (2.4%) RT sequences had a rearrangement in the 5' part of the RT, with an extensive molecular variation. The number of codons inserted between positions 68 and 69 ranged from 1 (3 samples) or 2 (41 samples) to 5 and 11 in one case each. In four cases, codon 67 was deleted. High levels of phenotypic resistance to zidovudine (AZT), lamivudine (3TC), stavudine (d4T), abacavir (ABC), and didanosine (ddI) were found in 95, 92, 72, 62, and 15% of the 40 samples analyzed, respectively. Resistance to AZT, d4T, and ABC could be found in the absence of the T215Y/F mutations. Resistance to 3TC could develop in the absence of specific mutations. Low-level resistance to ddI was noticed in 40% of the patients. The deletions of codon 67 seemed to have little effect on NRTI sensitivity. Most of the rearrangements were shown to contribute to cross-resistance to NRTI. The results regarding susceptibility to ddI raise the question of the interpretation of the phenotypic data concerning this drug.

JC virus genotypes in France: molecular epidemiology and potential significance for progressive multifocal leukoencephalopathy.

JC virus (JCV) induces progressive multifocal leukoencephalopathy (PML), especially in human immunodeficiency virus (HIV)-infected patients. Although JCV genotypes have primarily been associated with geographic patterns, a distinctive neuropathogenicity was recently attributed to genotype 2. A multicenter study was conducted to describe the distribution of JCV genotypes in France and to investigate correlations between genotypes and PML. Genotypes were determined by sequencing 494 bp in the VP1 capsid gene. Peripheral JCV was studied in 65 urine samples from 43 HIV-infected patients and from 22 control subjects. Genotypes 1, 4, 2, and 3 were detected in 52.3%, 30.8%, 12.3%, and 4.6% of the samples, respectively. In 56 brain or cerebrospinal fluid samples, PML-associated JCV of genotypes 1, 2, 4, and 3 was found in 66%, 19.7%, 8.9%, and 5.4%, respectively. Infection with JCV genotypes 1 or 2 was correlated with PML (odds ratio, 3.29). On the other hand, infection with JCV genotype 4 could represent a lower risk for PML.

Lessons from a multicentre study of the detectability of viral genomes based on a two-round quality control of GB virus C (GBV-C)/hepatitis G virus (HGV) polymerase chain reaction assay.

The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.

Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles.

[Does hepatitis c virus-related autoimmune thyroiditis exist? Reflections on a controlled study of 58 consecutive subjects]. / Les dysthyroïdies auto-immunes liées au virus C existent-elles? Réflexions à propos d'une étude contrôlée de 58 sujets consécutifs.

PURPOSE: Interferon alpha treatment for virus C hepatitis may be responsible for autoimmune thyroiditis. Relationships between thyroiditis and virus C infection are still debated. The aim of this study was to evaluate the existence of this association. METHODS: The prevalence of autoimmune thyroiditis in 58 patients (35 male and 23 female patients, mean age 52.6) with untreated virus C hepatitis was compared to that of 56 alcoholic patients (41 male and 15 female patients, mean age 53.8). Autoimmune thyroiditis was defined as the association of abnormal TSH and an increase in antithyroid antibodies. RESULTS: We did not find any statistical difference in either autoimmune thyroiditis or antithyroid antibodies prevalences. CONCLUSION: Both our results and a literature review suggest that the few reported cases of related autoimmune thyroiditis and virus C infection are probably coincidental.

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.

Detection of adenovirus in the waters of the Seine River estuary by nested-PCR.

Several systems for isolating viruses from environmental samples have been tested. The most promising method is based on genomic amplification. The authors attempted to detect adenovirus in nucleic-acid extracts from the Seine River estuary by a two-step amplification of a 220-bp segment of the conserved coding region of type 2 adenovirus hexon protein L3. The primers used in this study detected the most prevalent adenovirus serotypes in human disease in France, but not other virus strains or bacteria. The sensitivity of the nested polymerase chain reaction (PCR) amplification was estimated to be 10(2) copies of the adenovirus target sequence per ml of Seine River water. Nucleic-acid extracts from Seine River estuary waters were analysed and some tested positive for the presence of adenoviruses.

GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum.

PCR is, to date, the only available tool for the detection of GB virus C (GBV-C) and hepatitis G virus (HGV) RNAs. Twenty-two French laboratories participated in a quality control study to assess the sensitivity and specificity of their procedures. The panel included 13 positive controls and 7 negative controls. The laboratories used either in-house PCR techniques adapted from the literature or partly standardized commercial tests. Three laboratories performed faultlessly with the entire panel. Most laboratories had excellent specificity (100% in 20 of 22 laboratories). Sensitivity was acceptable (85 to 100%) in 15 centers and insufficient (38 to 77%) in 7. As with nonstandardized in-house PCR, the commercial assays gave discrepant performances in different laboratories. These results suggest that laboratories willing to use PCR for detection of GBV-C/HGV RNA for research or diagnostic purposes should participate in multicenter quality control trials.

Detection and genotyping of the hepatitis C RNA in tear fluid from patients with chronic hepatitis C.

Tear fluid from 51 patients with chronic hepatitis C virus (HCV) infection was analyzed for the presence of the hepatitis C RNA to assess the potential role of this fluid in virus transmission. HCV sequences were amplified from sera and tear fluids by nested polymerase chain reaction using primers from the 5' non coding region of the virus genome. Positive samples were genotyped by the LiPA procedures. HCV RNA was detected in 76.5% (39/51) of the sera and in 9.8% (5/51%) of the tear fluid samples. The presence of the RNA in the tear fluid was independent of the severity of the hepatitis and of the viral load as measured by the branched DNA assay. The genotypes of the tears and serum isolates were different for two patients. For another patient, the HCV RNA was positive in the tear sample but negative in the serum sample. These findings suggest that tear fluid may transmit HCV but the source of HCV RNA in this fluid needs to be better understood.

Hepatitis C virus infection in an HIV-positive population in Normandy: antibodies, HCV RNA and genotype prevalence.

The prevalence and the characteristics of hepatitis C virus infection (HCV) in 161 HIV-positive patients were studied. HCV seroprevalence was determined by enzyme immunoassay and recombinant immunoblot assay (RIBA). Two different reverse transcriptase polymerase chain reaction (RT-PCR) methods were also used to test the HCV-seropositive samples and 50 EIA-negative sera used as controls. The RNA HCV-positive sera were genotyped by the LiPA procedure. Associations of HCV status with demographic characteristics and risk factors were assessed by chi 2 and Fisher's exact tests. The seroprevalence of HCV was 34.2% with a significant difference between blood and sexual exposure risk groups (60.6% vs. 13.6%, respectively; P < 0.0001). Thirty-six of the 55 anti-HCV-positive sera were also positive for HCV RNA, and PCR detected HCV RNA in 8 HCV-seronegative patients. Various RIBA profiles were found and all sera were positive for antibodies to the c33 protein. A proportion of sera had elevated levels of transaminase activity (37.2%), and abnormal liver function as associated with HCV infection. Forty-two samples were genotyped and five genotypes and subtypes of the HCV virus were detected. Genotype 1a was the most frequent in this cohort, although genotype 1b is generally more common in France. The majority (94.1%) of the patients with genotype 1a had a history of blood exposure, which may account for the difference.

Herpes simplex hepatitis with chronic cholestasis in a newborn.

In the case of the central nervous system or hepatic involvement, the prognosis of neonatal herpes simplex infection remains poor, despite antiviral drugs, presumably effective if given early. We report the case of a neonate with herpes simplex hepatitis, where the course of the illness was unusual with chronic, ultimately fatal, cholestasis. The treatment was not effective, because its administration was delayed, because of high infant C reactive protein level and the absence of clinical maternal genital infection, and because it was interrupted due to misleading information: clinical improvement, negative viral tests and raised herpes IgG antibody titer.

HIV-1 sensitivity to zidovudine: a consensus culture technique validated by genotypic analysis of the reverse transcriptase.

In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.

Rapid decrease in the levels of the double-stranded RNA-dependent protein kinase during virus infections.

The double-stranded RNA-dependent protein kinase from human cells is a 68,000 molecular weight protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. With a monoclonal antibody specific for p68 kinase, here we show the phosphorylation and steady-state levels of p68 kinase during virus infection. The p68 kinase is phosphorylated in interferon-treated cells during infection with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and vaccinia virus, thus indicating activation of p68 kinase during these virus infections, an essential step required for autophosphorylation of p68 kinase. However, in spite of this activation, the level of p68 kinase is rapidly decreased in virus-infected cells. The half-life of p68 kinase in uninfected cells is 6 to 7 hr, whereas in EMCV-infected cells it is 2 to 3 hr. This decrease in the level of p68 kinase is dependent on the multiplicity of virus infection and it seems to be specific since other cellular proteins as well as the activity of 2'-5'-oligoadenylate synthetase are not modified. Decreased levels of p68 kinase are also observed in cells infected with VSV and vaccinia virus. In the absence of virus infection, decreased levels of p68 kinase occur in cells following incubation with poly(I).poly(C).

[Mycoplasmas and pregnancy]. / Mycoplasmes et grossesse.

The pathogenic role of mycoplasms during pregnancy remains quite controverted, depending on the studies; for some it has an incidence on prematurity, delayed growth in utero and premature rupture of the membranes. The purpose of this study was, from a population of patients with term delivery, without any specific pathology, to verify the frequency of mothers carrying Mycoplasma Hominis or ureaplasma, and to determine the possible consequences on the newborn. A linear analysis of the evolution of the samples between D0 and D6 in the mother and the new born, shows that the presence of mycoplasms in the genital passages is as frequent in this non-risk population, and that the child may be contaminated about every other time; but this contamination appears to be very transient and without any consequences on the immediate neo-natal pathology. Systematic screening of genital mycoplasms in pregnant women does not permit, therefore, to select a group of exposed patients. In newborns who are contaminated, the risk of infection appears to be very low, but it would perhaps be desirable to study the long range future evolution of healthy carriers.