A 1-year study of the epidemiology of hepatitis A virus in Tunisia.

This 1-year (September 2000 to August 2001) prospective study investigated the presence of hepatitis A virus (HAV) in the population of Monastir, Tunisia (86 serum samples), in the influents and effluents of two wastewater treatment plants, and in shellfish harvested in the coastal areas of Monastir, Bizerte and Sfax (January 2001 to May 2001). The virus was detected by RT-PCR using primers targeted at the VP3-VP1 region. An epidemic of HAV infection was observed during the winter months, with a peak in January. The presence of the virus was relatively constant in the influents and effluents of the wastewater treatment plants, and the virus was found in shellfish from the Monastir area during the months of January and February. The genotype IA strain was recovered most frequently from human serum and wastewater samples. The observation that the peak of the epidemic was during the winter months suggests that transmission of HAV is related to climatic factors and, presumably, to shellfish consumption.

A multiparametric flow cytometry method for detection of modifications of antigen expression in polymorphonuclear cells infected by human cytomegalovirus.

Human cytomegalovirus (CMV) has been shown to alter adhesion molecule expression on permissive cells such as endothelial cells. The aim of the present study was to investigate expression of receptors for these molecules on CMV infected polymorphonuclear leukocytes (PMNLs). CMV-induced variations on cellular integrin expression were examined using an in vitro system to obtain infected PMNLs. A triparametric flow cytometry approach was developed, which allows combined detection, in a single experiment, of both viral intranuclear antigen in the selected PMNLs and cellular CD11/CD18 expression. Comparison of infected PMNLs with uninfected cells showed a decrease of up to 50% in the expression of CD11b, CD11c, and CD18. This study thus demonstrates that the presence of CMV in PMNLs, which characterizes active infection, modifies the expression of integrins and may thus affect cell-to-cell interactions and immune functions.

Efficacy and safety of stavudine and didanosine combination therapy in antiretroviral-experienced patients.

OBJECTIVES: To assess the efficacy, tolerance, and safety of combination antiretroviral therapy with didanosine and stavudine in HIV-infected patients with CD4+ cell counts > 100 x 10(6)/l and HIV plasma RNA > 10(4) copies/ml previously treated with other antiretroviral agents for at least 3 months. DESIGN: In this open, multicentre, non-randomized, Phase II pilot study, adult patients were administered didanosine (200 mg twice daily) plus stavudine (40 mg twice daily) for 6 months. Patients for whom the first regimen had led to undetectable HIV RNA levels were offered a second 6-month course of treatment; those who had achieved insufficient immunological and virological gains in the first 6 months were given a new combination. METHODS: Primary evaluation of efficacy was based on viral load measured by branched DNA second-generation testing (lower limit of detection, 500 copies/ml) and CD4+ cell counts; secondary evaluations included AIDS-defining events and clinical side-effects. RESULTS: Sixty-five patients with median prior antiretroviral therapy of 24 months (65 with zidovudine, 29 with zalcitabine) were included in the study. At baseline, median CD4+ cell count was 198 x 10(6)/l and median plasma HIV RNA was 80000 copies/ml (4.9 log10 copies/ml). In this heavily pretreated population, an increase in the mean CD4+ cell count was observed (+70 x 10(6)/l at 24 weeks). In addition, rapid and prolonged antiviral activity was seen, with a mean maximal decrease of 1.1 log10 copies/ml at week 4, a mean decrease of 0.89 log10 copies/ml at week 24, and a plasma RNA viraemia < 500 copies/ml achieved in 14% of patients at week 24. CONCLUSIONS: Combination therapy with stavudine and didanosine is safe and leads to a sustained antiviral effect, even in patients with prolonged prior antiretroviral exposure and low CD4+ cell counts.

Usefulness of DNA viral load quantification for cytomegalovirus disease monitoring in renal and pancreas/renal transplant recipients.

BACKGROUND: The purpose of this prospective study was to evaluate the usefulness of quantifying DNA-cytomegalovirus (CMV) load for the diagnosis and monitoring of CMV disease among renal and pancreas transplant patients under immunosuppressive drugs. METHODS: A longitudinal study was conducted among 34 consecutive, unselected renal and pancreas/renal transplanted patients in our unit. During the first 3 posttransplant months, weekly monitoring of CMV infection and CMV disease was done, involving the determination of viremia by the shell vial assay, qualitative DNAemia by semi-nested polymerase chain reaction (PCR) and quantitative DNAemia by the hybrid capture system (HCS), a new and original hybridization method (337 samples were collected for each test). Qualitative and quantitative DNAemia results were blinded to physicians and three grades of disease were defined according to CMV related symptom occurrence. RESULTS: PCR was the most sensitive (100%) but the least specific (78%) method for the diagnosis of CMV disease. HCS was specific for CMV genome detection, sensitive and reproducible. Blood DNA levels above 60 pg/ml were predictive of severe or moderate CMV disease (sensitivity, 92%; specificity, 100%). A significant decrease in viral load was observed after ganciclovir administration, and a positive PCR or HCS result at the end of the antiviral treatment was associated with relapse of CMV infection or disease. CONCLUSIONS: It is concluded that quantitative DNAemia detection, with this new commercially available method, can predict disease and may be useful for a rational evaluation of ganciclovir preemptive therapy in such patients.

Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.

We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections.

BACKGROUND: Astroviruses (HAstVs) and 'Norwalk-like viruses' (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. OBJECTIVES: We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RT-PCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. STUDY DESIGN: All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25-40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. RESULTS: A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n=10), Bristol-like (n=8), and Hawaii-like viruses (n=1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. CONCLUSION: This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV.

Clinical and practical value of human cytomegalovirus DNAemia detection by semi-nested PCR for follow-up of BMT recipients.

The aim of this prospective study of 162 recipients of bone marrow transplantation (BMT) was to evaluate the use of DNAemia detection by semi-nested PCR for the diagnosis of human cytomegalovirus (HCMV) infection and HCMV disease. We compared the results obtained for DNAemia with those obtained for viremia, using the shell vial assay. Patients were divided in three groups, according to BMT type (allogeneic or autologous) and date of transplant; 876 DNAemia/viremia pairs were analyzed and the overall concordance between the two tests was 97.5%. Discrepancies between the two tests were essentially due to the earlier positivity of DNAemia. Among the 10 patients with positive DNAemia episodes, 9 developed HCMV disease. DNAemia was more sensitive than viremia for HCMV disease diagnosis, while viremia had a higher positive predictive value. DNAemia appeared easily adaptable to routine laboratory use, less expensive and more informative than viremia. This study shows that DNAemia is a method that can replace viremia detection, allowing HCMV infection and disease follow-up of recipients of allogeneic BMT during the first year after BMT.

Use of reverse transcription polymerase chain reaction with colorimetric plate hybridization to detect a cytomegalovirus late spliced mRNA in polymorphonuclear leukocytes from renal transplant patients.

Human cytomegalovirus replication was evaluated in polymorphonuclear leukocytes from ten renal transplant recipients. Three new reverse transcription polymerase chain reactions with plate hybridization suitable for automation were developed for the detection of immediate-early spliced UL123 mRNA, early-late pp65 mRNA, and late spliced UL22 mRNA. The presence of UL22mRNA was found to be significantly associated with the occurrence of cytomegalovirus (CMV) disease.

Colorimetric microtiter plate hybridization assay using monoclonal antibody for detection of an amplified human immunodeficiency virus target.

Since detection of amplified polymerase chain reaction products is a complicated procedure, a colorimetric microtiter plate hybridization assay was developed to automate specific detection of amplified HIV targets. In this assay, hybridization is seen by an antibody reacting selectively with double-stranded DNA. One hundred and ten amplified products detected by a DNA enzyme immunoassay (DEIA) and by classical hybridization with a digoxigenin-labeling probe, detection sensitivities were less than 10 HIV targets per 10(6) cells. This study demonstrates the specificity and sensitivity of DEIA for detecting amplified HIV targets. The use of the same equipment as for ELISA and the complete automation of the procedure allow a large number of samples to be processed in the clinical laboratory.

A one-step RT-PCR and a flow cytometry method as two specific tools for direct evaluation of human herpesvirus-6 replication.

In order to confirm the occurrence of active Human herpesvirus-6 (HHV-6) infection, two optimal procedures were developed to detect directly replicating virus. MT4 cells and peripheral blood mononuclear cells (PBMCs) infected with two different strains (HST and a patient strain GUI) were used. The first method consisted of a one-step reverse transcription PCR amplifying a part of the late alternatively spliced U100 gene which encode the gp 82-105 viral glycoprotein. Two extraction methods and two RT-PCR kits were evaluated, leading to the selection of TaKaRa mRNA selective PCR kit. The second procedure consisted in a flow cytometry method to analyze the expression of two late viral HHV-6 antigens using 7C7 and 10G6 monoclonal antibodies. Four fixation permeabilization procedures were compared and the preparation of cells with paraformaldehyde (PFA) 4% was found to be optimal. Evaluation of these methods was then realized during a sequential culture of HST strain on MT4 cells. This kinetic study confirmed that Mabs recognized late antigens and demonstrate that the U100 gene splicing starts at a late stage of multiplication whereas unspliced forms are detectable earlier in the cycle.

Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification.

When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. Seven techniques for extracting hepatitis A virus (HAV) from stool and shellfish samples were compared, in order to identify the protocol most suited to both types of sample and with the best extraction yield. The protocols tested were either techniques for the recovery and purification of total RNA, such as RNAzol, PEG-CETAB, GTC-silica and Chelex, or techniques for isolating specifically HAV using a nucleotide probe or a monoclonal antibody. For stool samples, RNAzol, PEG-CETAB, and magnetic beads with antibody allowed detection of the virus in 11/12 and 12/12 of samples. For shellfish samples, three protocols allowed RNA to be extracted in 90% of cases, RNAzol, PEG-CETAB, and GTC-silica. Their rapidity and low cost make RNAzol and GTC-silica the most suitable for routine diagnostic testing. reserved.

Detection of human papillomavirus in squamous intraepithelial lesions by consensus and type-specific polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify human papillomavirus (HPV) in 216 cervical biopsy specimens from women referred to the gynecological out-patient unit for colposcopy because of an abnormal smear. HPV DNA was screened using type-specific primers for HPV6, 11, 16, 18, 31 and 33 (TS-PCR) as well as a consensus primer located in the E1 region of the HPV genome (C-PCR). TS-PCR specificity was validated by Southern blot analysis. Low-grade (SIL 1) and high-grade (SIL 2) squamous intraepithelial lesions were found in 165 biopsies. HPV16 detection was better with PCR than Southern blot, particularly for SIL 1 and SIL 2. The fact that 10% of HPV16 (all SIL 2) were not detected by C-PCR indicates that both PCR techniques should be performed. C-PCR also detects uncharacterized HPV types (8.6% prevalence in our results), mainly in SIL 1 and SIL 2. HPV16, the most frequently isolated type (prevalence 21%), was associated with SIL 2 in 83% of cases. A low HPV prevalence was found in specimens without dysplastic cells. These results suggest that PCR may be an important tool for identifying women at risk for developing dysplasia or cervical cancer.

[Epidemiology of Horton's disease. Is there an environmental factor, infective in particular?]. / Epidémiologie de la maladie de Horton. Existe-t-il un facteur d'environnement, en particulier infectieux?

Epidemiological studies of temporal arteritis have essentially only been reported in the English literature. The authors of this study were concerned with this aspect of temporal arteritis in the Loire-Atlantique region of France over a period of 10 years (1970-1979). The high prevalence in white races has been confirmed. The annual incidence in France is comparable to that seen in Northern Europe and the USA. The incidence of the disease is especially high between 70 and 80 years. The apparent female predominance is related to the greater life expectancy in women. The study of several conjugal cases does not suggest the intervention of an infectious agent. The same is true for isolated cases where the responsibility of a bacterial or viral agent has not been demonstrated. Other environmental factors (sun exposure, life-style, socio-professional classification) do not affect the incidence of the disease. The genetic background would seem to be of particular importance. This study found a significantly higher prevalence of HLA DR4 antigen, confirming the results of American and British studies. However, in contrast to previous studies, this series did not confirm an increase in HLA B8 antigen.

[Microgallery for the identification of anaerobic bacteria]. / Utilisation d'une microgalerie pour l'identification des bactéries anaérobies.

A microgallery for the identification of anaerobic bacteria (API 20A) was used with regard to 213 strictly anaerobic strains isolated between 1.12.74 and 30.11.75 in a hospital laboratory. Furthermore, 75 strains of Bacteroides fragilis isolated previously were added to this study. All the strains were identified by traditional methods. The results of tests performed with the microgallery alone permitted the species diagnosis in 72,2% of cases. Performing simultaneously complementary tests (7 to 9 Tubes, according to the nature of the bacteria) the species diagnosis was possible in 89.9% of cases. The main interest of this microgallery was its use for the identification of glucidolytic strains for it included the presence of 17 ternary substances. Its use for the other groups of strains was thus limited.

[Study of the transferable factors of antibiotic resistance in the enterobacteria isolated at the Regional Hospital Center of Nantes]. / Etude des caractères transférables de la résistance aux antibiotiques chez les entérobactéries isolées au Centre Hospitalier Régional de Nantes

Characteristics of transferable resistance of 72 entero-bacteriaceae resistant to one more antibiotic, isolated from February to September 1972, showed that this was common. Multi-resistant strains permit one to obtain transfer of various characteristics either alone or together in 76 per cent of cases, whereas strains with less than three characteristics, transferred in only 9 per cent of cases. The transfers were uncommon in the case of Proteus compared with other enterobacteriaceae and, in particular, Klebsiella, Escherichia coli and Salmonella.

[Comparison of 2 immunoenzyme methods for the determination of anti-cytomegalovirus IgM]. / Comparaison de deux méthodes immunoenzymatiques pour la détermination des IgM anti-cytomegalovirus.

An antibody capture assay technique using a labeled antigen (Wellcome Laboratories) and an ELISA technique performed after elimination of serum IgG (Behring Laboratories) were assessed for determination of anti-cytomegalovirus IgM. Their specificity and sensitivity were compared in tests on 208 sera, some (60) of which had IgM rheumatoid factor, antinuclear antibodies or anti-Epstein-Barr virus antibodies. The other sera were analyzed relative to diagnosis of a cytomegalovirus infection. The antibody capture assay technique was slightly more sensitive than the ELISA technique but did not resolve all the specificity problems.

[Typing of papillomaviruses in cervical dysplasias. Its value in treatment]. / Typage des papillomavirus dans les dysplasies du col. Intérêt pour leur traitement.

The typing fo Human Papillomavirus (HPV) was carried out in cervical lesions in order to decide on the best therapy to carry out in mild and medium dysplasias of the cervix. 131 patients who had an iodo-negative zone or a smear suggesting an HPV infection had microbiopsies carried out under colposcopic control. Every lesion had two biopsies carried out side by side to study the histopathology and the virology. Dysplastic lesions were found in 93 biopsies. A search for the DNA of HPV 6a, 16 and 18 was carried out using a Southern blot technique with 32P following the method of Random multipriming. Hybridization was carried out in strict and non-strict conditions. In 43 cases out of 93 dysplasias a virus was found: 2 HPV 6a, 14 HPV 16, 1 HPV 18 and 26 X HPV's (not typed). The number of HPV's that could be detected increased according to the severity of the dysplasia which was also found in frequency of type 16. Where the biopsies showed no dysplasia type 16 was found in four out of seven viruses. In this study electrophoretic profiles for type 16 were found in four cases suggesting that they were integrated into the chromosome of the cell. The low percentage of HPV found in mild or moderate dysplasias (4 type 16 out of 61 biopsies) shows that at present this test can not be used as an aid in choosing treatment.

[Antibacterial effects of the combinations of azlocillin with other antibiotics]. / Effets antibactériens des associations d'azlocilline avec d'autres antibiotiques.

Evaluating the synergistic effects of antibiotic associations is by no means an easy task owing to the diversity of the test methods utilized for this purpose. Data extracted from 11 reports published between 1975 and 1982 and concerning associations of azlocillin with various aminoglycosides (gentamicin, sisomycin, netilmicin, amikacin, tobramycin and dibekacin) and with two beta-lactam antibiotics (cloxacillin and cefotaxime) are reviewed in this article. On the whole, a synergistic effect against Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacteriaceae was most frequently observed with the azlocillin-aminoglycoside association. This effect was usually more pronounced with organisms showing low sensitivity to azlocillin and less pronounced with organisms resistant to aminoglycosides. As often pointed out, there is no general rule that would help predict with certainty from the sensitivity of a strain whether or not any given antibiotic association will act synergistically on that strain.

[Search of papillomavirus genome in synovial membranes and rheumatoid nodules]. / Recherche de génome de papillomavirus dans les synoviales et les nodules rhumatoïdes.

IgG antibodies to rat esophagus stratum corneum are highly specific for rheumatoid arthritis and have been recently shown to be directed against human (pro)filaggrin. Although the mechanisms underlying the loss of tolerance of B lymphocytes for filaggrin remain unelucidated, a role of infection with the human papillomavirus has been suggested on the basis of the following facts: 1) the protein exhibiting the greatest molecular homology with filaggrin is papillomavirus-encoded; 2) human papillomavirus proteins bind to cytoskeletal proteins; 3) human papillomavirus replication is enhanced by friction and rheumatoid lesions predominantly develop in areas subjected to friction, as shown by the distribution of rheumatoid nodules; 4) the rheumatoid pannus resembles a benign tumor. The objectives of this study were to look for the human papillomavirus genome in six synovial membrane specimens and two rheumatoid nodule specimens from rheumatoid arthritis patients with disease durations of more than ten years and in 20 synovial membrane specimens collected during surgical procedures in osteoarthritis patients. The methods used were polymerase chain reaction with papillomavirus-specific primers. The papillomavirus genome was not detected in any of the 28 specimens. A role for the human papillomavirus in the pathogenesis of rheumatoid arthritis cannot be ruled out on the basis of these findings because filaggrin is expressed in other tissues, including the thymic medulla epithelium. In addition, the primers used in this study may have been inappropriate.