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KEY MESSAGE: We propose new methods to predict genotype × environment interaction by selecting relevant environmental covariates and using an AMMI decomposition of the interaction. Farmers are asked to produce more efficiently and to reduce their inputs in the context of climate change. They have to face more and more limiting factors that can combine in numerous stress scenarios. One solution to this challenge is to develop varieties adapted to specific environmental stress scenarios. For this, plant breeders can use genomic predictions coupled with environmental characterization to identify promising combinations of genes in relation to stress covariates. One way to do it is to take into account the genetic similarity between varieties and the similarity between environments within a mixed model framework. Molecular markers and environmental covariates (EC) can be used to estimate relevant covariance matrices. In the present study, based on a multi-environment trial of 220 European elite winter bread wheat (Triticum aestivum L.) varieties phenotyped in 42 environments, we compared reference regression models potentially including ECs, and proposed alternative models to increase prediction accuracy. We showed that selecting a subset of ECs, and estimating covariance matrices using an AMMI decomposition to benefit from the information brought by the phenotypic records of the training set are promising approaches to better predict genotype-by-environment interactions (G × E). We found that using a different kinship for the main genetic effect and the G × E effect increased prediction accuracy. Our study also demonstrates that integrative stress indexes simulated by crop growth models are more efficient to capture G × E than climatic covariates.

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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.

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A cooperative study was conducted among six laboratories to compare the performance of the Cobas Amplicor (CA) polymerase chain reaction (PCR) system (Roche Molecular Systems, USA) for the detection of Mycobacterium tuberculosis with that of microscopy and culture in routine clinical laboratory diagnosis. A total of 5,221 decontaminated respiratory specimens were tested. The use of an internal control allowed detection of PCR inhibition in 144 (2.8%) specimens. Only two culture-positive samples were CA PCR inhibitory and therefore could not be detected by PCR testing. Of the 333 culture-positive specimens, 278 (83.5%) were positive by the CA PCR. Of the 4,744 culture-negative specimens, 52 (1.1%) were positive by the CA PCR. After analysis of discrepancies, 40 of the 52 culture-negative, CA PCR-positive specimens were classified as true positive. Thus, the overall sensitivities of culture, CA PCR and microscopy were 89.3%, 85.2% and 55.5%, respectively. The overall specificity of the CA PCR was 99.7%. Five of the six centers found similar performances for the CA PCR, with sensitivities ranging from 85.7 to 90.9%. The CA PCR was more sensitive for smear-positive samples, exhibiting overall sensitivities of 96.1% and 71.7% for smear-positive and smear-negative specimens, respectively. These results indicate that the Cobas Amplicor system enables microbiology laboratories with reasonable previous experience in molecular biology testing to perform PCR and to detect Mycobacterium tuberculosis in more than 70% of specimens obtained from infected patients.

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The Amplicor HCV Monitor test for quantitative determination of serum or plasma hepatitis C virus (HCV) RNA was modified recently and introduced onto the Cobas Amplicor instrument to automate fully amplification, detection and calculation of results. This new version (v2.0) was evaluated in a routine diagnostic laboratory. The sensitivity and reproducibility were assessed on well-characterized panels (Eurohep) and clinical samples. HCV RNA levels measured by the v2.0 Monitor test were about 1log(10) higher than those detected by the previous version test, and genotypes 1 and 3 were quantified with equal sensitivity. Within the linear dynamic range of 10(3) to 10(6) copies/ml, the coefficients of variation for both intra- and inter-assay reproducibility ranged from 1.9 to 2.95%. This test system was found to be a reliable and labor saving assay for the quantification of HCV RNA.

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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.

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The aim of this study was to investigate the following in a large population of French patients with chronic hepatitis C: the geographical distribution of hepatitis C virus (HCV) genotypes; the relationship between HCV genotypes and epidemiological characteristics; severity of the disease; and response to interferon (IFN) therapy. Data from 14 tertiary referral centres, corresponding to 1872 patients with chronic hepatitis C, were prospectively collected from 1989 to 1997. HCV genotyping was performed using the line probe assay (LiPA). HCV genotypes 1b, 3, 1a, 2, 4 and a mixed infection were found in 41%, 22%, 16%, 11%, 4% and 4% of our population, respectively. HCV genotype distribution was homogeneous, except for genotype 2 that was found more frequently in the southwest than in the other regions (21% vs 9.2%) (P=0.001). HCV distribution was associated with gender, age, and source and duration of infection. In multivariate analysis, these correlations were related to the source of infection, which was the only independent factor significantly associated with genotype (P=0.001). Genotype 1b was significantly more common in patients with cirrhosis, but in multivariate analysis cirrhosis was independently related to older age at exposure and longer duration of infection (P=0.001). A sustained response to IFN therapy was observed in 11% of patients infected with genotypes 1a or 1b vs 32% of those infected with genotypes 2 or 3 (P=0.001). This study shows that HCV genotype is mainly related to the source infection, but not to the intrinsic pathogenicity of HCV, and is a strong predictor of sustained response to therapy.

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The fully automated COBAS AMPLICOR CT/NG test for the detection of Chlamydia trachomatis was evaluated in a multicenter trial. Test performance was evaluated for 2,014 endocervical swab and 1,278 urine specimens obtained from women and for 373 urethral swab and 254 urine specimens obtained from men. Culture served as the reference test. Culture-negative, COBAS AMPLICOR-positive specimens that tested positive in a confirmatory PCR test for an alternative target sequence within the C. trachomatis major outer membrane protein gene were resolved as true positives. The overall prevalence of chlamydia was 4.3% in cervical swabs and 11.0% in urethral swabs from men. When the results for each specimen type were considered separately, the resolved sensitivities were 96.5% (83 of 86) for endocervical swab specimens, 95.1% (39 of 41) for urine specimens from women, 100.0% (41 of 41) for urethral swab specimens from men, and 94.4% (17 of 18) for urine specimens from men; the resolved specificities were 99.4% (1,912 of 1,924) for endocervical swab specimens, 99.8% (1,204 of 1,207) for urine specimens from women, 98. 5% (325 of 330) for urethral swab specimens from men, and 100.0% (236 of 236) for urine specimens from men. For the subset of patients from whom both swab and urine specimens were collected, the combined results for both specimen types were used to identify all infected patients. Using these combined reslts as criteria, the resolved sensitivities for the COBAS AMPLICOR test were 82.6% (38 of 46) for endocervical swab specimens, 84.4% (38 of 45) for urine specimens from women, 84.2% (16 of 19) for urethral swab specimens from men, and 89.5% (17 of 19) for urine specimens from men. In comparison, the sensitivity of culture was only 56.5% (26 of 46) for endocervical specimens and 63.2% (12 of 19) for urethral specimens from men. The internal control provided in the COBAS AMPLICOR test revealed that 2.9% of specimens were inhibitory when they were initially tested. Nevertheless, valid results were obtained for 99. 1% of specimens because 68.7% of the inhibitory specimens were not inhibitory when a second aliquot of the original sample was tested. Two additional COBAS AMPLICOR-positive specimens were detected by retesting inhibitory specimens. The COBAS AMPLICOR CT/NG test for the detection of C. trachomatis exhibited equally high sensitivities and specificities with both urogenital swab and urine specimens and, thus, is well-suited for use in screening.

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Morbidity resulting from the toxicity of chemotherapeutic drugs suggests that novel approaches are worthy of investigation. Development of the use of low intensity magnetic fields as an adjuvant to current treatment regimens to prevent metastatic disease may prove to be efficacious. Using a cell culture model, we have developed a magnetic field (MF) treatment that offers the possibility of lowering the therapeutic dose of these drugs and thereby reducing morbidity. Our studies have found that a low intensity (approximately 2 gauss) MF signal and a relatively low dose (0.1 microg/ml) of Adriamycin (ADR) inhibited proliferation of human osteosarcoma cells by 82%, whereas the MF and ADR acting individually caused only 19% and 44% inhibition, respectively.

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METHODS: Test-of-cure of 19 patients with Chlamydia trachomatis genital infection was assessed by daily collection of first void urine for 7 days just after treatment by azithromycin single-dose. RESULTS: Detection by PCR and TMA of C. trachomatis showed a good correlation between both methods. The observation that post-therapy chlamydial nucleic acid detection is associated to bacterial clearance suggests that all the patients were cured.

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The benefits shown by the recent introduction of PCR for the in vitro diagnosis of hepatitis C virus (HCV) infection has prompted the development of standardized, ready-to-use assays that can be implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV (COBAS), the first instrument system that allows the automation of HCV RNA amplification and detection, to determine its performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the results were compared with the results for biochemical and serological markers of HCV. In this study the two PCR systems showed the same accuracy, with a concordance rate of 99.8%. As expected, the correlation between serology and PCR was not absolute because the presence of anti-HCV antibodies may be associated with a latent or past infection. On the other hand, if the presence of confirmed anti-HCV antibodies and elevated alanine aminotransferase levels are taken as the "gold standard," indicating an active, ongoing infection, the COBAS and AMPLICOR tests show high and comparable sensitivities (100%) and specificities (98%), with positive and negative predictive values of 100 and 97%, respectively. During the study no false-positive reactions were detected. The use of an internal control allowed the identification of inhibitory substances that prevented amplification for 0.3 and 0.4% of samples tested by the COBAS and AMPLICOR tests, respectively. Compared to the manual system, the COBAS system allowed a significant reduction of hands-on time and could improve the overall laboratory work flow. In conclusion, these results support the use of the COBAS and AMPLICOR tests for the molecular diagnosis of active HCV infections.

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BACKGROUND: Vasculitides are diseases of unknown origin in the majority of cases, but sometimes are the consequence of viral infections; for instance, hepatitis B virus (HBV)-related polyarteritis nodosa (PAN) or hepatitis C virus (HCV)-associated cryoglobulinaemia. OBJECTIVE: To investigate the role of hepatitis G or GB virus C (GBV-C) in various forms of medium- and small-vessel vasculitides. DESIGN: Retrospective analyses of sera. SETTING: Tertiary care hospital in Bobigny, France. PATIENTS: Fifty-six vasculitides: 19 HBV-PAN, 10 PAN without HBV infection, 11 microscopic polyangiitis (MPA), seven Churg-Strauss syndrome (CSS) and nine Wegener's granulomatosis (WG). Every sample was collected before treatment. MEASUREMENTS: GBV-C RNA was detected using a reverse transcription-polymerase chain reaction assay with primers derived from the conserved GBV-C helicase and NS5a regions. RESULTS: GBV-C was detected in five of the 56 samples (8.9%): four patients with HBV-related PAN and one with MPA; three of these patients (two with HBV-PAN, one with MPA) had been transfused and two HBV-PAN were i.v. drug addicts. GBV-C was not found in CSS or in WG. CONCLUSION: GBV-C infection was observed only in patients who had been transfused or who were addicts. This virus is unlikely to have a primary role in vasculitides.

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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.

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Multicentre quality control studies demonstrated that optimization and standardization of HCV RNA reverse transcription polymerase chain reaction (RT-PCR) amplification techniques were possible: thus, a nested HCV RNA RT-PCR assay was described as a reliable tool for the detection of hepatitis C viral RNA. Besides this, another procedure, the Amplicor HCV RNA qualitative test, a standardized RT-PCR assay, became available. In order to assess the relationship between seropositivity and potential infectivity and to compare both RT-PCR assays, all patients undergoing maintenance hemodialysis were evaluated at hospital de Meaux (Meaux, France) during one year. We conclude that both assays are equally sensitive and that acid guanidinium thiocyanate based methods, which are used to prepare RNA prior to PCR, are more efficient than the usual phenol extraction protocols. Care should be taken with sera from hemodialyzed patients as the presence of inhibitors of the PCR has been demonstrated during the course of HCV RNA testing.

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Third-generation recombinant immunoblot assay is widely used for the validation of the serological diagnosis of hepatitis C virus infection. To determine whether indeterminate recombinant immunoblot assay 3.0 patterns may be associated with viral replication and liver disease, 89 indeterminate patterns were studied (67 c22n 14 c33c, 5 c100p and 3 NS5); 35 (39%) had immunosuppression. Serum alanine aminotransferase activity was increased in 49 (55%); HCV RNA was evidenced through polymerase chain reaction in 52 (58%). The observation of indeterminate recombinant immunoblot assay 3.0 justifies investigation of liver disease and search for HCV RNA, since a large proportion of individuals with such patterns are hepatitis C virus-infected.

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Blood tonometry is the only method to assess the accuracy of the pO2 determination on blood gas analyzers. ABL instruments by Radiometer were tested by two types of tonometry (film and bubble tonometry) and the validity of the algorithm for pO2 correction was analysed with these results. The role of the presentation of the specimen is also discussed. For the precision study on the ABL 510 analyzer, coefficients of variation for pO2 were < 0.37% and < 1.7% for within-run and day-to-day series respectively. pO2 accuracy was excellent. Linearity was verified between 0-620 mmHg (82.5 kPa), and interinstrument comparisons demonstrated a strict correlation with the Ciba-Corning 178 instrument. ABL 510 measures simultaneously oxygen saturation by spectrophotometry. Analytical results showed an acceptable level of imprecision, but the definition and the clinical significance of this parameter are ambiguous.

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Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

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Coproporphyrinogen oxidase was purified to homogeneity from mouse liver. The specific activity of the pure enzyme was 3500 nmol.h-1.mg-1; its apparent molecular mass (35 kDa) was confirmed by immunological characterization of the enzyme in a trichloroacetic-acid-precipitated total-liver-protein extract. The native enzyme appeared to be a dimer of 70 kDa as determined by gel filtration under nondenaturating conditions. The Km value for coproporphyrinogen III was 0.3 microM. The purified enzyme was activated by neutral detergents and phospholipids (affecting both Vmax and Km) but inhibited by ionic detergents. Reactivity toward sulfhydryl agents suggested the possible involvement of (an) SH group(s) for the activity. When compared to the previously purified coproporphyrinogen oxidases (from bovine liver and yeast), the mouse liver coproporphyrinogen oxidase appears to share many common catalytic properties with both enzymes. However, its apparent molecular mass is very different from that of the bovine liver enzyme (71.6 kDa) but identical to that found for the yeast (Saccharomyces cerevisiae) enzyme.

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We have investigated the regulation of ferritin synthesis during induction of Friend erythroleukemic cells by dimethyl sulfoxide. Northern blot analysis shows that mouse ferritin H and L mRNAs each contain approximately 1.1 kilobases. The levels of both mRNAs increase after addition of dimethyl sulfoxide in a biphasic manner. After a sharp rise in the first 6 h, the levels decline and then rise again over the next 90 h. These increases in mRNA levels reflect increased transcription of both mRNAs. Analysis of ferritin subunit synthesis surprisingly showed no corresponding increase in the rate of protein synthesis, suggesting that the additional mRNA was not in functional polysomes. These studies also indicated a novel processing of mouse ferritin H subunits. H subunits appear to be synthesized as a precursor of approximately 22,500. This form is not present in mature shells. Pulse-chase experiments indicated that the precursor is first processed to an intermediate form of 20,000 and then to the 18,000 component found in functional shells.

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Porphobilinogen deaminase is induced during the dimethyl sulfoxide-mediated differentiation of Friend erythroleukemia cells. We have previously shown that when succinylacetone, a potent inhibitor of porphobilinogen formation, is present during the differentiation process, the induction of the enzyme is apparently suppressed. Here, we provide evidence that, in this condition, porphobilinogen deaminase is synthesized normally but does not accumulate as a consequence of an accelerated turnover. The normal half-life of the protein is 24 h but decreases to 10 h when the formation of its substrate is impaired by succinylacetone. We propose that when the enzyme is covalently bound to its substrate, a normal step in this enzymatic reaction, it is protected from proteolytic degradation, and we show that this new finding is relevant to the human disorder acute intermittent porphyria.

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