[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.

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.

Plasma protein kinase activity enhanced by interferon is found in platelets.

A protein kinase activity analogous to that found in interferon-treated HeLa cells is detectable in human plasma rich in platelets. This kinase activity is manifested by the phosphorylation of an endogenous Mr 72000 protein which could be conveniently assayed after partial purification on poly(G)-Sepharose. Here, we show that the protein kinase system in the plasma consists of at least 2 components. The protein kinase is found to be localised in the platelets whereas most of the substrate (the Mr 72000 protein) is found free in the plasma and a fraction of it associated with the surface of platelets.

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.

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.

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.

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.

[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.

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.

Enzyme markers for the presence of circulating interferon: 2-5A synthetase in blood lymphocytes and protein kinase in platelet-rich plasma.

The level of 2-5A synthetase in extracts of peripheral blood lymphocytes and a specific protein kinase activity in platelet-rich plasma were measured in normal individuals and in patients suffering from viral or bacterial infections. The level of these enzymes was tested at different times during the disease. The level of 2-5A synthetase and the protein kinase activity was enhanced by several-fold during viral and bacterial infections and decreased during the course of the disease in parallel with clinical ameliorations and reversal of clinical symptoms. Among the different types of infections studied, higher levels of these enzymes were observed during viral than bacterial infections. Our results emphasize the use of these enzymes as markers to evaluate the state of the disease and recovery. Furthermore, they provide evidence for the production of interferon during different types of infection.

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.

[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.

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.

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).

Assay and the levels of 2-5A-synthetase in lymphocytes of patients with viral, bacterial and autoimmune diseases.

The level of 2-5A-synthetase in extracts of peripheral blood lymphocytes was estimated by the capacity of the enzyme to synthesize 2-5A in the presence of ATP. The 2-5A was then purified on a small column of DEAE-cellulose. Here we show that under or experimental conditions, the amount of 2-5A formed is proportional to the level of the enzyme. The concentration of 2-5A (nmol of AMP/10(6) lymphocytes) in an assay therefore reflects the level of the enzyme. Enhanced levels of 2-5A synthetase were observed in the lymphocytes of patients with viral and bacterial infections. In most of the cases studied, these enhanced levels of the enzyme decreased during the course of infection parallel to recovery. Thus, the level of 2-5A synthetase may indicate the state of the disease and its evolution during the period of treatment; furthermore, it may be a useful marker in monitoring the return to a normal physiological condition. In addition to patients with viral and bacterial infections, enhanced levels of 2-5A synthetase were observed in patients suffering from autoimmune diseases. In five patients studied here, the enhanced levels of 2-5A synthetase remained high at different periods during the course of the disease. These results suggest the presence of circulating interferon throughout the disease.

Continuous production of interferon in normal mice: effect of anti-interferon globulin, sex, age, strain and environment on the levels of 2-5A synthetase and p67K kinase.

Two interferon-mediated enzymes, a 2-5A synthetase and a kinase that phosphorylates a 67 000 mol. wt. (p67K) protein were found at variable levels in different organs of mice. Among the different strains of mice included in this study, germ-free mice had the lowest levels of these enzymes. The levels of 2-5A synthetase and p67K kinase were enhanced significantly in all mice following treatment with mouse (alpha + beta) interferon. Here, we show that the presence of 2-5A synthetase and p67K kinase in different organs of normal mice (untreated) was due, at least in part, to a constant production of interferon under different physiological conditions. Accordingly, injection of normal mice with anti-mouse interferon (alpha + beta) globulin led to a significant decrease in the level of 2-5A synthetase and p67K kinase. In conventional mice (C3H/He), the level of both of these enzymes was higher in female than in male animals and was decreased with age or when such animals were reared isolated in a pathogen-free protected unit. The levels of 2-5A synthetase and p67K kinase were also decreased in normal mice following injection with a powerful antibiotic against a very wide spectrum of Gram-positive and Gram-negative bacteria. These results suggest that the production of interferon was induced continuously in normal mice. Such induction was mediated by both internal and external agents.

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.

Enhanced level of double-stranded RNA-dependent protein kinase in peripheral blood mononuclear cells of patients with viral infections.

The protein kinase activity dependent on double-stranded RNA was assayed in extracts of peripheral blood mononuclear (PBM) cells from healthy volunteers and in patients with different types of virus infections. The protein kinase was assayed after one-step purification on an immunoaffinity column containing monoclonal antibody against the 68,000 Mr protein, a subunit of the protein kinase. In healthy individuals, the activity of the protein kinase remains constant. In contrast, the activity of the protein kinase is enhanced significantly in patients with viral infections and is decreased during the course of the disease in parallel with clinical ameliorations and reversal of clinical symptoms. There is a strong correlation between the enhanced levels of the protein kinase activity and another interferon-mediated enzyme, 2-5A synthetase. Both of these enzymes, therefore, could be used as markers to evaluate the state of the disease and recovery. In the different populations of lymphocytes, most of the protein kinase activity was found to be present in T and B lymphocytes, T lymphocytes showing a higher activity than B lymphocytes.