Progressive multifocal leukoencephalopathy despite immune recovery in a HIV/HCV co-infected patient.

In HIV patients, HCV co-infection has been associated with an increased risk of progressive multifocal leukoencephalopathy (PML). Furthermore, PML has also been described in patients with cirrhosis, whether related to HCV infection or not. We describe here the case of a HIV/HCV co-infected patient with cirrhosis who developed PML despite HIV suppression and CD4 cell count above 250/mm3 for 2 years. Immunological studies performed at onset of PML and before HCV therapy showed a decrease in naïve CD4 cells (CD45RA+CCR7+CD27+ CD4+ T cells - 23% cells, i.e. 75/mm3) and NK lymphopenia with abnormal and activated NK cells (CD3- CD16+ and/or CD56+) (5% lymphocytes, i.e. 58/mm3, CD69 91%, NKp30 26%). This impaired immunity, possibly related to HIV infection, or HCV infection or cirrhosis, or a combination thereof, could have led to the development of PML.

Severe acute hepatitis E in an HIV infected patient: Successful treatment with ribavirin.

In industrialized countries, most cases of hepatitis E virus (HEV) infection in humans are autochthonous, mainly through foodborne and zoonotic transmission routes. In Europe, genotype 3 is a cause of acute self-limiting viral hepatitis, but can also be responsible for chronic hepatitis in immunocompromised patients. Ribavirin has been successfully used in the treatment of chronic hepatitis E and in a few cases of severe acute hepatitis E in immunocompetent patients. We report here the case of a 39 year-old man infected with HIV presenting with acute hepatitis E (genotype 3c). Unlike most cases, evolution was severe with a fall of prothrombin time down to 45%. Treatment with ribavirin allowed rapid viral clearance and a gradual normalization of liver function tests.

[Epstein-Barr virus and cytomegalovirus primary infections: a comparative study in 52 immunocompetent adults]. / Étude comparative des primo-infections à Epstein-Barr virus et à cytomégalovirus chez 52 patients immunocompétents.

OBJECTIVE: The objective of this study was to compare epidemiological, clinical, and biological data of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) primary infections in immunocompetent adults, admitted in the infectious disease department of the Reims Teaching Hospital between 2000 and 2005. PATIENTS AND METHODS: Inclusion criteria were the presence of anti-VCA IgM antibodies or the presence of CMV specific IgM antibodies and the absence of any other positive serology. Differences in reported percentage were compared with a Khi(2) test or Fischer's exact test, when appropriate. Continuous variables were compared with the Mann-Whitney Test. RESULTS: There were no significant changes over the years in the numbers of EBV (n=32) and CMV (n=20) primary infections. The patient's mean age was 22.7 years (14-48 years) in EBV primary infections and 38.6 years (13-66 years) in CMV primary infections (P<0.01). The clinical variables significantly associated with primary EBV infection were sore throat and cervical lymphadenopathy (P<0.01). Arthromyalgia and respiratory manifestations were less frequent in EBV primary infection (P<0.01). The biological variables significantly associated with EBV primary infection were a marked alanine aminotransferase elevation and a marked lymphocytosis with atypical lymphocytes (P<0.001). Thrombopenia was less frequently associated with EBV primary infection (P<0.001). CONCLUSION: Clinical and biological presentations of EBV and CMV primary infections were similar. The simultaneous serologic diagnosis of these two infections remains necessary to provide a specific diagnosis, for the most efficient patient care.

Nephropathia epidemica and leptospirosis in Champagne-Ardenne, France: comparison of clinical, biological and epidemiological profiles.

In the present retrospective study, we described a series of 45 non-icteric leptospirosis and 44 nephropathia epidemica (NE) patients diagnosed in the northeast of France from 1995 to 2005 and compared their clinical picture and laboratory parameters, as well as some epidemiological data. Loin pain (P < 0.001), abdominal pains (P = 0.007), rise of blood pressure (P < 0.001) and pharyngitis (P = 0.01) were more frequently found in NE patients. Aspartate aminotransferase (ASAT) (P = 0,006), creatine phosphokinase (CPK) (P < 0.0001) and C-reactive protein (CRP) (P < 0.0001) were higher in leptospirosis, whereas creatinine (P = 0.009) was higher in NE. Leptospirosis mainly concerns occupational hazards, e.g. farmers, and leisure activities like swimming, and NE concerns professional foresters or leisure activities in the forest and the cleaning of attics. During hospitalisation, patients receiving antibiotics were more frequent among leptospirosis than among NE patients (80% versus 59%, P = 0.06). Among the various common clinical signs, only acute myopia appeared to be a pathognomonic but inconsistently observed clinical sign, which was only observed in 47% of NE cases.

[A case of influenza A community-acquired pneumonia in an elderly subject hospitalised in intensive care unit]. / A propos d'un cas de pneumopathie communautaire grippale chez un sujet âgé en réanimation.

Influenza pneumonia and influenza-associated severe exacerbation of pre-existing heart and lung disease are responsible for major complications that may require intensive care unit admission. Here, we report the case of a diabetic 70 year-old man hospitalised in the intensive care unit (ICU) of the University Medical Center of Reims (France) for a severe bilateral and alveolar pneumonia requiring mechanical ventilation. This patient had received a classical antibiotic treatment by amoxycillin (3 g/24 hours per os); 48 hours later, he was admitted in ICU for a respiratory failure that evolved rapidly towards an acute respiratory distress syndrome. Because of the context of a winter influenza outbreak, a nasal swabbing sample was tested for the presence of Influenzavirus nucleocapsid-antigens (Immunochromatographic test; BinaxNow Flu A & B, Binax, Portland, USA). This rapid assay revealed the presence of an Influenzavirus A respiratory infection five days after the beginning of the respiratory syndrome. This rapid viral diagnosis will be further confirmed in post mortem by the positive Influenza strain isolation onto lung tissues by classical cell culture techniques (Influenzavirus A strain, H3N2). Influenza pneumonia is a significant cause of morbidity and mortality, especially during influenza epidemics. The use of commercially available rapid diagnostic tests for influenza associated pneumonia, allows the potential use of new specific anti-neuraminidase drugs, which can be efficient during the 30 hours after the beginning of the clinical influenza syndrome.

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.

[A fatal case of Herpes simplex virus meningoencephalitis in an immunocompetent adult]. / Un cas mortel de méningoencéphalite à Herpes simplex virus chez un adulte immunocompétent.

Management of herpes simplex virus encephalitis (HSE) has been considerably improved by the development of rapid polymerase chain reaction (PCR) assays and by the use of intravenous acyclovir. However, an absence of early antiviral treatment has been associated to a poor outcome in patients with HSE. In the present report, we described the case of a 53 years-old adult immunompetent patient who was admitted to the emergency department of university medical center of Reims (France). At the time of hospitalisation, he was suffering from a febrile encephalitis syndrome evolving for more than 24 hours. A cerebrospinal fluid (CSF) puncture was performed demonstrating the presence of a lymphocytic meningitidis (42 leukocytes/mm3 which 90% of mononuclear cells; CSF protein = 1650 mg/L) associated with high levels of interferon alpha (75 UI/mL). Specific herpesvirus PCR and hybridisation assays (Herpes Consensus Hybridowell, Argene, France) were positive for the detection of HSV-1 genome on this CSF sample. Despite the intravenous acyclovir treatment (15 mg/kg/8 hours) delivered at the time of hospitalisation, this immunocompetent adult patient will dead 15 days later by a cardiorespiratory failure that was related to extensive HSE lesions. The time delay between the beginning of the clinical syndrome and the instauration of intravenous acyclovir treatment (more than 24 hours) was the only point susceptible to explain the presence of extensive CNS lesions in this patient. Specific Herpesvirus PCR detection assays are powerful tools that are actually used to establish a rapid etiological diagnosis of viral meningo-encephalitis. However, in patients demonstrating clinical signs of encephalitis associated with an aseptic CSF, it remains essential to urgently initiate a presumptive intravenous acyclovir treatment (10-15 mg/kg/8 hours). Actually, this medical practice is the only one susceptible to reduce the morbi-mortality rates linked to HSE.

Economic impact of healthcare-associated rotavirus infection in a paediatric hospital.

The aim of this prospective study was to determine the extra-length of stay and the average cost for rotavirus healthcare-associated infection (HAI). Children admitted to the paediatric ward of the Reims University Hospital between the 1 December 2001 and 31 March 2002, were included in a pairwise matched (1:1) case-control study. Cases were defined as patients with rotavirus HAI. Controls were selected according to matching variables in a stepwise fashion. The costs measured in this study included all expenses sustained by the hospital. Information on costs was obtained from medical records and the hospital economic department. The attack rate and the incidence of healthcare-associated acquired rotavirus infection were 6.6% and 15.8 per 1000 hospital days, respectively, during a winter outbreak. Fifteen percent of HAI were identified after discharge. The average cost per case was 1930 and the mean excess length of stay was 4.9 days. Our findings clearly demonstrate the substantial expense incurred as a result of HAI caused by rotavirus in children. To prevent these costly infections, several cost-effective measures such as standard precautions should be reinforced in the education of the healthcare workers.

Virological and histological responses to one year alpha-interferon-2a in hemodialyzed patients with chronic hepatitis C.

BACKGROUND: alpha-Interferon-2a (IFNalpha) alone is a therapy of limited proven benefit for non-uremic patients with chronic hepatitis C virus (HCV) infection. In dialyzed patients, such an effect is suggested on small short-term studies without sufficient clinical and virologic follow-up to document any sustained effect. PROTOCOL: Twelve chronically hemodialyzed patients with chronic hepatitis C and waiting for renal transplantation were included in a prospective open study of treatment with IFNalpha. We used, as did others, doses of 3 million units (MU), three times a week, but for a longer period of treatment of 12 months. Follow-up was continued for 6 months after the end of IFNalpha in order to document any sustained biochemical, virological and histological responses. RESULTS: Aminotransferase levels returned to the normal range within 1-2 months of treatment in all patients in whom they had been elevated at baseline. At 1 month of treatment, serum HCV-RNA was not detected in 5 (41%) patients and in 9 (75%) at 12 months. A sustained virological response was documented in 4 (33%) patients 6 months after the end of treatment. Relapse occurred in 5 patients within 2 months after IFNalpha withdrawal. HCV genotype was not predictive of any sustained response. At inclusion, using the histologic Metavir scoring system, half of the patients had low-grade cytolytic activity and none had cirrhosis. After IFNalpha, liver biopsy specimens were available from 9 patients and showed histologic improvement in 3. IFNalpha tolerance was poor, inducing a 5% mean weight loss and the acute rejection of two nonfunctioning kidney grafts. CONCLUSION: This study documents that administration of IFNalpha at 3 MU three times a week, for 12 months, in hemodialysis patients with chronic hepatitis C was efficient for clearing the serum of HCV-RNA in 75% of the patients. A sustained response was maintained in one third of these patients after cessation of IFNalpha, and was predicted by the early serum clearance of the virus within the first 2 months of treatment. We confirm that a 12-month treatment period carries a higher sustained response rate than shorter treatment periods. These encouraging results call for larger studies in uremic patients, using IFNalpha alone or in association with new antiviral drugs.

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

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

Detection of specific immunoglobulin E during maternal, fetal, and congenital toxoplasmosis.

Toxoplasma immunoglobulin E (IgE) antibodies in 664 serum samples were evaluated by using an immunocapture method with a suspension of tachyzoites prepared in the laboratory in order to evaluate its usefulness in the diagnosis of acute Toxoplasma gondii infection during pregnancy, congenital infection, and progressive toxoplasmosis. IgE antibodies were never detected in sera from seronegative women, from patients with chronic toxoplasma infection, or from infants without congenital toxoplasmosis. In contrast, they were detected in 86.6% of patients with toxoplasmic seroconversion, and compared with IgA and IgM, the short kinetics of IgE was useful to date the infection precisely. For the diagnosis of congenital toxoplasmosis, specific IgE detected was less frequently than IgM or IgA (25 versus 67.3%), but its detection during follow-up of children may be interesting, reflecting an immunological rebound. Finally, IgE was detected early and persisted longer in progressive toxoplasmosis with cervical adenopathies, so it was also a good marker of the evolution of toxoplasma infection.

Characterization of Dac g 4, a major basic allergen from Dactylis glomerata pollen.

Monoclonal antibodies were produced against Dac g 4, a purified major basic allergen from Dactylis glomerata pollen. Their ability to be used for immunopurification of Dac g 4 was studied on a BIAcore apparatus (Pharmacia). The allergen was purified by affinity chromatography with one monoclonal antibody. Its precise molecular mass, 59,185 +/- 30 d, was determined by mass spectrometry. Its isoelectric point is 10.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting showed that Dac g 4-related proteins of similar molecular mass were detected in the majority of allergenic grass pollen species. By double-site ELISAs, we have estimated that Dac g 4 represents about 6% of the total proteins from a water-soluble extract. One monoclonal antibody (mAb H) recognized a 60 kd cross-reactive protein in other grass pollens, though none in any of the tree or weed pollens tested. Inhibition studies of IgE antibody binding to Dac g 4 with pollen extracts confirmed the presence of cross-reactive allergens in Secale cereale, Lolium perenne, Festuca elatior, Holcus lanatus, Bromus arvensis, Poa pratense, Hordeum sativum, and Phleum pratense.

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins.

BACKGROUND: Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. OBJECTIVE: We sought to characterize the allergen biochemically and immunologically. METHODS: The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. RESULTS: The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. CONCLUSIONS: Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

Comparison of three horizontal two-dimensional electrophoretic techniques to separate grass pollen allergens.

The identification and characterization of allergenic components are important for improving both diagnosis and therapy of allergy. We have studied grass pollen crude extract for better characterization of the allergen repertoire recognized by allergic patient IgE antibodies. Two-dimensional electrophoresis are the methods of choice to define physico-chemical characteristics of the allergens as they give isoelectric points and molecular mass of the analyzed samples. We compared the advantages and the drawbacks of three different ways to perform a two-dimensional electrophoresis. First, two gel media were compared to run native isoelectric focusing (IEF) with carrier ampholytes: agarose and acrylamide. With the introduction of immobilized pH gradient, highly resolutive and reproducible 2D electrophoresis could be obtained. This technique was also applied to separate the allergens of Dactylis glomerata pollen.

DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples.

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

Two-dimensional gel electrophoretic analysis with immobilized pH gradients of Dactylis glomerata pollen allergens.

We studied Orchard grass pollen allergens (Dactylis glomerata) by a new two-dimensional electrophoretic technique which is the combination of isoelectric focusing (IEF) in immobilized pH gradient (IPG) and horizontal SDS-PAGE. The IPG-Dalt (two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension) electrophoresis was made with four different but complementary pH gradients: 3-10.5, 4-9, 4-7, 6.3-10.5. We also compared the behavior of allergens in native and denaturing conditions during IEF. This technique followed by electroblotting on nitrocellulose membrane and immunodetection with an allergic patient serum was shown to increase the number of IgE-binding components up to 100 compared to one-dimensional techniques. Major allergen groups could be identified. New allergens not yet described could be characterized by their isoelectric points and their molecular masses. This highly resolving and reproducible technique, should allow an easy allergen standardization.

Modified silver staining for immobilized pH gradients.

Silver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to absorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micromolar level, to decrease the background.