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.

Surveillance network for herpes simplex virus resistance to antiviral drugs: 3-year follow-up.

Herpes simplex virus (HSV) infections are very common in the general population and among immunocompromised patients. Acyclovir (ACV) is an effective treatment which is widely used. We deemed it essential to conduct a wide and coordinated survey of the emergence of ACV-resistant HSV strains. We have formed a network of 15 virology laboratories which have isolated and identified, between May 1999 and April 2002, HSV type 1 (HSV-1) and HSV-2 strains among hospitalized subjects. The sensitivity of each isolate to ACV was evaluated by a colorimetric test (C. Danve, F. Morfin, D. Thouvenot, and M. Aymard, J. Virol. Methods 105:207-217, 2002). During this study, 3900 isolated strains among 3357 patients were collected; 55% of the patients were immunocompetent. Only six immunocompetent patients excreted ACV-resistant HSV strains (0.32%), including one female patient not treated with ACV who was infected primary by an ACV-resistant strain. Among the 54 immunocompromised patients from whom ACV-resistant HSV strains were isolated (3.5%), the bone marrow transplantation patients showed the highest prevalence of resistance (10.9%), whereas among patients infected by human immunodeficiency virus, the prevalence was 4.2%. In 38% of the cases, the patients who excreted the ACV-resistant strains were treated with foscarnet (PFA), and 61% of them developed resistance to PFA. The collection of a large number of isolates enabled an evaluation of the prevalence of resistance of HSV strains to antiviral drugs to be made. This prevalence has remained stable over the last 10 years, as much among immunocompetent patients as among immunocompromised patients.

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.

Genetic analysis of hepatitis A virus outbreak in France confirms the co-circulation of subgenotypes Ia, Ib and reveals a new genetic lineage.

Genetic analysis of selected genome regions of hepatitis A Virus (HAV) suggested that distinct genotype could be defined in different geographic locations. In order to study the degree of genetic variability among HAV isolated during a single epidemic outbreak, sequences from a 148 base pair segment within the VP1 amino terminal region were obtained for eight distinct HAV isolates from an outbreak that occurred in North Bretagne (France). These sequences were compared among themselves and with published sequences from 30 different strains that represented different HAV sub-genotypes that were isolated all over the world. Phylogenetic analysis revealed an extensive genetic heterogeneity among strains belonging to the same outbreak and revealed co-circulation of sub-genotype IA, IB, and the presence of IIIA sub-genotype for the first time in a Mediterranean country.

Human herpesvirus 6 infection after autologous or allogeneic stem cell transplantation: a single-center prospective longitudinal study of 92 patients.

To determine the incidence and clinical relevance of active human herpesvirus 6 (HHV-6) infection, 92 consecutive unselected recipients of autologous or allogeneic stem cell grafts were investigated in a prospective longitudinal study. Active infection was assessed by the presence of viral deoxyribonucleic acid (DNA) in 846 peripheral blood mononuclear cell specimens and 115 plasma specimens, by means of a specially developed polymerase chain reaction designed to avoid detection of latent genome. The incidence of HHV-6 infection observed was 42.5%, irrespective of the type or source of graft, and infection was significantly associated with partial (P=.002) or total myelosuppression (P=.01) and fever (P<. 000001). Infusion of bone marrow as the source of graft, reactivation occurring before platelet or neutrophil engraftment, and presence of HHV-6 DNA in plasma were identified as risk factors for symptomatic HHV-6 infection (P<.002).

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 relevance of direct quantification of pp65 antigenemia using flow cytometry in solid organ and stem cell transplant recipients.

A total of 1,305 blood samples from 85 solid organ transplant (SOT) recipients and 25 stem cell transplant (SCT) recipients at risk for cytomegalovirus (CMV) infection were prospectively collected and tested using the shell vial assay (SVA) and a leukocytic qualitative PCR (q-PCR). Of these, 462 specimens were further tested by direct quantification of CMV antigenemia by flow cytometry (FC-Ag), 125 were tested with a quantitative competitive PCR, and 200 were tested for pp65 antigenemia using the slide method (S-Ag). Laboratory data were statistically analyzed according to the presence of CMV-related symptoms. In SOT and SCT recipients, active CMV infection occurred in 63.5 and 36%, respectively, and CMV disease occurred in 53 and 24%, respectively. FC-Ag results correlated better with q-PCR and S-Ag than with SVA. The first test found to be positive during follow-up was FC-Ag in 73% of cases. In SOT recipients, FC-Ag showed the highest sensitivity and negative predictive value for the diagnosis of any grade of CMV disease. For FC-Ag, the threshold beyond which CMV disease was highly probable seemed to lie at 0.20% positive polymorphonuclear leukocytes. FC-Ag appears to be a useful test for the early detection of CMV infection and the prediction of CMV disease.

Sequential use of paraformaldehyde and methanol as optimal conditions for the direct quantification of ZEBRA and rta antigens by flow cytometry.

A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.

Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry.

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.

Increased anti-Epstein-Barr virus antibodies in epidermotropic cutaneous T-cell lymphoma: a study of 64 patients.

Epstein-Barr virus (EBV) is often associated with non-Hodgkin's T-cell lymphomas and has recently been found in the lesions of mycosis fungoides and Sézary syndrome. We sought to determine whether the anti-EBV antibody profile was disturbed in mycosis fungoides and Sézary syndrome and whether there are particular profiles characteristic of disease stage. Anti-EBV antibodies (anti-VCA, -EA and -EBNA) were studied in the sera of 64 patients. An immunoenzymatic technique was used, and the results were compared with the same number of age- and sex-matched healthy controls. Patients with mycosis fungoides and Sézary syndrome developed higher anti-VCA antibody titres (median 1200) than controls (median 320). Thirty-seven patients had anti-VCA > or = 1200 vs. 19 controls (P < 0.01). These elevated anti-VCA antibody titres were associated with positive EA in 19 patients versus three controls. No differences were found between the illness stages. Anti-EBV antibodies were most often found in mycosis fungoides and Sézary syndrome when the serological profile was similar to that of cellular immune deficiencies and EBV-related non-Hodgkin's lymphoma. EBV could be involved, either directly on lymphocytes or, more likely, indirectly by chronic antigenic stimulation.

Direct sequencing of hepatitis A virus strains isolated during an epidemic in France.

Direct sequencing of PCR products was used to study the VP1 region of the hepatitis A virus (HAV) genome (position 2199 to 2356) of nine strains isolated from human stools collected during a hepatitis A epidemic (western France, 1992), three strains from environmental samples (1990, 1991, and 1992), and two HAV cell culture isolates (the French strain CF53/Lyon and strain CLF). These viruses differed from CF53/Lyon (genotype I) by between 1 and 10.3%, and results indicated the existence of two groups of strains belonging to two different subgenotypes (IA and IB). With this sequencing technique it was possible to monitor the epidemiology of HAV and study its relations.

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.

[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.

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.

[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.

[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.

[Critical study of the serological diagnosis of herpetic encephalitis. Apropos of 4 cases]. / Etude critique du diagnostic sérologique de l'encéphalite herpétique. A propos de quatre observations.

Virologic study was performed in four cases of Herpes simplex virus encephalitis. Herpes simplex virus type 1 was recovered from surgical brain biopsy specimens taken in three cases. Anti-HSV antibodies were assayed in simultaneous blood and cerebrospinal fluid samples by ELISA. CSF and serum specific antibody ratios were compared to those of antibodies directed against other viral antigens whenever possible. A significant and early increase in this CSF/serum ratio was demonstrated in two patients. The diagnostic value of this serological method is discussed, as an alternative to viral isolation from brain biopsy specimens.

[Serial infections with two herpesviruses after renal transplantation: virological and ultrastructural features (author's transl)]. / Infection séquentielle à deux herpèsvirus après une transplantation rénale: aspects virologiques et ultrastructuraux.

A fatal case of active viral infection in a transplant patient is reported. On the second month post transplantation, Cytomegalovirus (CMV) infection occurred. It was attested by clinical and radiological symptoms, transient increase of transaminases and seroconversion with CMV antigen. Patient apparently recovered, but on the third month mucocutaneous Herpesvirus hominis (HVH) infection started. It was rapidly complicated by hepatitis and encephalitis which are directly responsible for the fatal outcome. A strain of HVH type 1 was easily isolated from oral and genital lesions. Immunofluorescence with HVH antiserum was positive on post mortem sample of liver biopsy Ultrastructural changes and intranuclear Herpesvirus like particles were demonstrated in liver. Etiology of hepatitis and management of immunosuppressive therapy are discussed.