Tracheal amylase dosage as a marker for microaspiration: a pilot study.

BACKGROUND: Devices that limit microaspiration through the cuffs of endotracheal tubes could help prevent ventilator-associated pneumonia (VAP). The amount of tracheal microaspirations could be a relevant study endpoint. The aim of our study was to assess whether amylase measured in tracheal secretions constituted a relevant marker for microaspiration. METHODS: Twenty-six patients, intubated for at least 48 h and supplied with a subglottic secretion-suctioning device, constituted a group with a high risk of microaspiration. Twelve non-ventilated patients that required a bronchoscopy procedure constituted a group with a low risk of microaspiration (the control group). Tracheal (T) amylase was compared between the groups. In the intubated group, a series of oral (O), subglottic (Sg) and tracheal (T) suction samples were collected and T/O, T/Sg, Sg/O amylase ratios were determined. RESULTS: Amylase was measured in 277 (89 Sg, 96 B, 92 T) samples from the intubated group and in 12 T samples from the control group. Tracheal amylase was lower in the control group than the intubated group (191 [10-917] vs. 6661 [2774-19,358] IU/L, P<0.001). Amylase gradually increased from tracheal (6661 [2774-19,358] IU/L), to subglottic (130,750 [55,257-157,717] IU/L), to oral samples (307,606 [200,725-461,300] IU/L), resulting in a median 5.5% T/O ratio. In a subset of intubated patients, T amylase samples were assessed in two different laboratories, and gave reproducible results. CONCLUSION: Tracheal amylase was easy to collect, transport, and measure. The T/O amylase ratio is a first step towards quantifying oropharyngeal to tracheal microaspiration in mechanically-ventilated patients.

[Extensive bone marrow necrosis with sepsis in primary thrombocytemia: a case report and literature review]. / Nécrose médullaire avec sepsis compliquant une thrombocytémie essentielle: à propos d'une observation et revue de la littérature.

INTRODUCTION: Bone marrow necrosis is a very rare condition which is characterized by a necrosis of hematopoietic progenitors, adipocytes and reticulin network. CASE REPORT: We report a 62-year-old woman admitted to an intensive care unit for an essential thrombocytemia associated with bone marrow necrosis complicated by septic shock and progressive multi-organ failure. To our knowledge, this is the second case reported in the literature. The clinical presentation of bone marrow necrosis includes non-specific symptoms such as fever, bone pain and sometimes a clinically significant medullar insufficiency syndrome. Biology can reveal cytopenias, elevated LDH and alkaline phosphatase serum levels. The diagnosis is confirmed by bone marrow trephine biopsy. Bone marrow necrosis is classified as extensive if more than 50% of the bone marrow biopsy show necrosis. Haematological malignancies (particularly leukaemia), and solid malignant tumours (particularly gastro-intestinal or lung cancers) represent up to 90% of aetiologies and must be actively researched. Also, sickle cell disease and catastrophic anti-phospholipid syndrome must also be investigated. Essential thrombocytemia remains an exceptional cause of bone marrow necrosis. CONCLUSION: Overall the prognosis of bone marrow necrosis is poor unless appropriate and intensive treatment, especially for sickle cell disease in which complete medullar regeneration has been observed.

[Gelatinous transformation of the bone marrow and acute hepatitis in a woman suffering from anorexia nervosa]. / Thrombopénie par transformation gélatineuse de la moelle et hépatite aiguë de dénutrition chez une patiente anorexique mentale.

Anorexia nervosa can be a life-threatening eating disorder when complicated with electrolyte disturbance, gelatinous transformation of the bone marrow or starvation induced acute hepatitis. We report a 43-year-old woman suffering from anorexia nervosa for more than 25 years, who was admitted in intensive care unit for a fluctuating level of consciousness related to starvation-induced acute hepatitis. Gelatinous transformation of the bone marrow was also diagnosed. Those two entities are rare and, to our knowledge, have not been previously reported jointly in anorexia nervosa.

Rotation thromboelastometry detects thrombocytopenia and hypofibrinogenaemia during orthotopic liver transplantation.

BACKGROUND: Orthotopic liver transplantation can be associated with haemorrhage, particularly in patients with severe liver dysfunction. We assessed the value of rotation thromboelastometry (ROTEM) to monitor coagulation in the operating theatre, its correlation with routine laboratory findings, and its ability to guide platelet (Plt) and fibrinogen (Fg) transfusion. METHODS: Twenty-three patients were included in this prospective observational study. Laboratory tests and ROTEM tests (EXTEM, INTEM, FIBTEM, and APTEM) were performed six times during the procedure. Correlations between laboratory findings and ROTEM parameters were sought. Thresholds for ROTEM parameters were determined with receiver-operating characteristic (ROC) curve analysis according to Plt count and Fg levels. RESULTS: Clot amplitude at 10 min (A10) of EXTEM was well correlated with Plt count and Fg levels (R(2)=0.46 and 0.52, respectively, P<0.0001). FIBTEM A10 was correlated with Fg (R(2)=0.55, P<0.0001). ROC analysis showed that EXTEM A10 with a threshold of 29 mm predicted thrombocytopenia with a sensitivity of 79% and a specificity of 60%, and a threshold of 26 mm predicted hypofibrinogenaemia with a sensitivity of 83% and a specificity of 75%. CONCLUSIONS: ROTEM is useful for the global assessment of coagulation in the operating theatre. EXTEM was the most informative for assessing the whole coagulation process and A10 showed value in guiding Plt and Fg transfusion.

Grafted self-assembled monolayers derived from naturally occurring phenolic lipids.

Self-assembled monolayers grafted onto silicon surfaces were obtained from the hydrosilylation products by trialcoxysilanes of naturally occurring phenolic lipid allyl ethers. The as-obtained materials were characterized by various physical and physicochemical methods. Thus, contact angles of water drops showed that they possess very high hydrophobicity. Their excellent regularity was corroborated by AFM microscopy. The frequencies of the stretching CH2 infrared modes indicate the presence of alkyl chains mainly in the trans/trans conformation. Additionally, optical ellipsometry and quartz microbalance measurements enabled us to estimate the thickness of the films. The results, as a whole, are in good agreement with the formation of densely packed monolayers.

Identification and characterization of the neutralization epitope(s) of the hepatitis E virus.

The neutralization epitope(s) of the hepatitis E virus (HEV) was studied by an in vitro neutralization assay using antibodies obtained by immunization of mice with 51 overlapping 30-mer synthetic peptides spanning the region 221-660 amino acids (aa) of the HEV open reading frame 2 encoded protein (pORF2) and 31 overlapping recombinant proteins of different sizes derived from the entire pORF2 of the HEV Burma strain. Antibodies against synthetic peptides and short recombinant proteins of approximately 100 aa did not neutralize HEV, suggesting the HEV neutralization epitope(s) is conformation-dependent. However, one recombinant protein of approximately 400 aa in length comprising the pORF2 sequence at position 274-660 aa as well as all truncated derivatives of this protein containing region 452-617 aa elicited antibodies, demonstrating HEV neutralizing activity. These findings establish for the first time that the minimal size fragment, designated pB166, that can efficiently model the neutralization epitope(s) is 166 aa in length and is located at position 452-617 aa of the HEV pORF2. Additionally, antibodies against pB166 were found to cross-neutralize three different HEV genotypes, suggesting that a common neutralization epitope(s) may exist within the different HEV genotypes. Thus, recombinant proteins constructed in this study may be considered as potential candidates for the development of an HEV subunit vaccine as well as for the development of highly sensitive and specific diagnostic tests.

[Psychorelational aspects of the end of life]. / Aspects psychorelationnels de la fin de la vie.

The patient nearing the end of life should be considered as a living being who is suffering and who has needs. Overall the suffering is physical, psychological and spiritual. The patient undergoes this final existential crisis filled with both anxiety and hope. Each person has unique mechanisms of defense and adaptation, linked to his personality and background. These unique reactions must be recognised and taken into account in care and in communication in order to maintain a living relationship to the end.

Evidence of hepatitis E virus replication on cell cultures.

Several human and animal cell lines have been used to grow hepatitis E virus. The strain SAR-55 was adapted only on PLF/PLC/5 cell line without any visible cytopathic effect. The growth of the SAR-55 was monitored by examining the positive and the negative strands of HEV-RNA. Stool samples, obtained from hospitalised acute hepatitis patients at the Fever Hospital of Alexandria (Egypt), were used to confirm the susceptibility of PLF/PLC/5 cells. After more than one-week's cultivation, three stool samples out of 17 IgM anti-HEV positive and 1 from 52 IgG anti-HEV positive patients showed a specific RT-PCR amplification product. The nucleotide sequences of the methyltransferase region of the genome in the isolates revealed the maximum homology with Burma strain with several point mutations.

Primary structure of open reading frame 2 and 3 of the hepatitis E virus isolated from Morocco.

The nucleotide sequence from position 5,014 to 7,186 of the hepatitis E virus (HEV) genome was determined using a set of 10 polymerase chain reaction (PCR) fragments amplified directly from a pool of fecal specimens obtained from patients with well-documented epidemic HEV infection in Morocco. This sequence contains the 3'-terminal region of open reading frame 1 (ORF1), full length ORF2 and ORF3, and a portion of the 3'-noncoding region. The HEV Morocco nucleotide sequence was compared with the corresponding sequences of 13 HEV strains. A region of ORF2 that overlaps with ORF3 was found to be the most conserved region of ORF2, whereas a protein segment encoded by this region was found to be the most variable. Theoretical RNA secondary structure analysis predicted that this region may be folded into a strong secondary structure that may constrain nucleotide sequence variability. In addition, the nucleotide sequence comparison revealed that the HEV Morocco sequence is most homologous to the sequences of the HEV Asian strains compared with the HEV Mexico, swine, and US strains. Phylogenetic analysis performed on the entire ORF2 and ORF3 sequences and on a small fragment of ORF2 allowed classification of the HEV Morocco strain together with a few other known African strains as a separate subtype within the Asian-African genotype.

Neutralization of different geographic strains of the hepatitis E virus with anti-hepatitis E virus-positive serum samples obtained from different sources.

A recently developed polymerase chain reaction (PCR)-based cell culture neutralization assay was used to investigate cross-neutralization of known hepatitis E virus (HEV) strains obtained from various HEV-endemic regions of the world with different anti-HEV-positive serum samples. Serum specimens obtained from cynomolgus macaques experimentally infected with strains from Burma, Mexico, or Pakistan cross-neutralized the infectivity of each strain as well as an isolate from Morocco. Serum samples obtained either from infected patients who reside in HEV-endemic regions of the world or from U.S. residents who became infected while traveling to such regions also neutralized all four strains. In contrast, antibodies obtained from rabbits immunized with full-length Burma strain ORF2 protein neutralized only the Burma and Pakistan strains, not the Mexico or Morocco strains. In addition, antibodies obtained from guinea pigs immunized with an N-terminal truncated Burma strain ORF2 protein neutralized each strain except the Morocco strain. These data strongly suggest that antibodies elicited during an HEV infection demonstrate broad HEV neutralizing activity, whereas antibodies elicited after immunization with recombinant Burma ORF2 protein demonstrate a more limited ability to neutralize various HEV strains obtained from different regions of the world endemic for the disease.

At least three epitopes are recognized by the human repertoire in the hepatitis B virus group a antigen inducing protection; possible consequences for seroprevention and serodiagnosis.

Many recent investigations have shown that both HBV and anti-HBs antibodies coexist in the same patient, and HBV can be found in individuals with anti-HBc antibodies in the absence of immunologically detectable HBsAg. In most cases, mutant forms of HBV affecting the region of the envelope gene coding for the group a determinant recognized by human antibodies have been found. The nature of the group a determinant was revisited with an ELISA involving dissociated, but not alkylated, envelope subunits. No antibody recognizing a continuous epitope of the major S envelope protein could be found in humans; the full activity of human anti-HBs antibodies appeared to be focused on the discontinuous group a determinant. The immunological human repertoire against the HBsAg group a determinant was analysed by competitive inhibition of three mouse monoclonal antibodies (mAbs) selected as recognizing three distinct specificities on the group a determinant. Antibodies to specificities #1 and 3 were found in 52/70 anti-HBs human sera and generally predominated over specificity #2, which was lacking in some sera. The heterogeneity of the group a determinant suggested by these data argue for the use of more than one type of anti-HBs mAb for seroprevention of recurrence after liver transplantation and HBs serodiagnosis. Provided all three types of mAbs characterized here recognize HBV variants with mutations in the a determinant and are virus-neutralizing, it may be helpful, after association of such mAbs, to use them for diagnosis and to devise new immunotherapeutic strategies to prevent emergence of HBsAg escape mutants.

High-level ability of secretory IgA to block HIV type 1 transcytosis: contrasting secretory IgA and IgG responses to glycoprotein 160.

The IgG and secretory IgA (S-IgA) responses to the HIV-1 envelope (gp160 antigen) were analyzed in the colostrum (Col) and in the cervicovaginal fluid (CVF) of HIV-l-infected women. We show IgG antibodies (Abs) to the recombinant gp160 to be predominant as compared with the corresponding S-IgA isotype. The low level of the S-IgA response cannot be related to a general disturbance of the mucosal-associated Iymphoid tissue (MALT) because the level of a current Ab to a caries-associated antigen from Streptococcus sobrinus was in the normal range in these secretions. The major subclass of IgA to gp160 was of the alpha1 isotype both in Col and in CVF. However, the specific activities of S-IgA1 and S-IgA2 were different when expressed as the ratio of the anti-gp160 related to total Ig of each subclass. Indeed, the specific activity of the S-IgA2 was predominant over S-IgA1 in the Col, whereas the reciprocal results were found in CVF, showing a subcompartmentalization of these secretions. The ability of S-IgA and IgG to block one of the pathways involved in the HIV-1 penetration across mucosa, i.e., transcytosis through epithelial cells, was evaluated using a functional in vitro assay. Both S-IgA and IgG Abs impaired virus transcytosis, irrespective of the level of antigp160 specific activities. However, specific S-IgA was more efficient than IgG. These features suggest that mucosal specific S-IgA to HIV-1 could be relevant in decreasing infectivity of HIV-1 in corporal fluids.

African strains of hepatitis E virus that are distinct from Asian strains.

Partial genomic sequences of four hepatitis E virus (HEV) strains from Africa (Morocco and Tunisia) and one from Central Asia (Tashkent, Uzbekistan) were obtained. The reverse transcriptase-polymerase chain reaction was used to amplify 5' and hypervariable regions of open reading frame 1 (ORF1) and a region overlapping all 3 ORFs. Sequence analysis of these regions revealed the African strains to be quite distinct from all known Asian strains but more similar to them than to the Mexican strain. Sequence analysis of the Tashkent strain revealed almost complete identity with another central Asian strain from Osh, Kirgizia. These results thus further confirm the geographical origin of HEV strain divergence.

Selective unresponsiveness to HBsAg vaccine in newborns related with an in utero passage of hepatitis B virus DNA.

Thirty four out of 158 (22%) newborns to mothers chronically infected by the hepatitis B virus (HBV) did not produce antibodies (Ab) to HBsAg 1 month after the last injection of the HBV vaccine supplemented with HBV specific immunoglobulins. At birth, HBV genome was detected by polymerase chain reaction (PCR) in the peripheral blood mononuclear cells (PBMC) of a large majority (28 out of 34) of these non-responder newborns but never in the other newborns who responded to the HBsAg vaccine. HBV genome was detected in serum, only in some cases (nine out of 34) and never in the absence of HBV DNA in PBMC. For nine out of 14 followed newborns, the absence of response was transitory since anti-HBs Abs appeared after 15 months, without booster, while the HBV genome had disappeared. Unresponsiveness was specific to the HBV envelope protein since all late responders and 15-months-non-responders to the HBsAg vaccine produced normal levels of Abs to the three poliovirus serotypes, to tetanus toxoid and to the pneumococcus polysaccharides. An in utero induced immune tolerance to low doses of HBsAg appears as the most plausible hypothesis to explain this unresponsiveness to HBV vaccine.

A new PCR-based seroneutralization assay in cell culture for diagnosis of hepatitis E.

A new method for the serological diagnosis of hepatitis E virus (HEV) infection based on neutralization of the virus in cell culture was developed. The test involves a short incubation of the virus in the presence of the serum sample to be tested and permissive cells. With viral replication being limited and without a cytopathic effect, viral growth in cells is evaluated by reverse transcription and PCR. The specificity of the test was established by studying sera from healthy individuals and patients with hepatitis living in France, where autochthonous hepatitis E is unknown. The kinetics and sensitivity of antibody detection were evaluated during the experimental infection of monkeys. Neutralizing antibodies were found in 79% of patients during an outbreak of hepatitis E and in 43% of patients with sporadic, acute non-A, non-B (without anti-hepatitic C virus antibodies) hepatitis. This neutralization assay is proposed as a confirmatory test for the available enzyme-linked immunosorbent assay (ELISA), which is now recognized as giving many false-positive reactions, and to improve identification of new hepatitis viruses since false-negative reactions with HEV ELISA are also encountered.

New quantitative assay of hepatitis B and C viruses by competitive PCR using alternative internal sequences.

A competitive PCR was developed for quantitation of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA, alternatively, using only two constructions containing both priming sites. DNAs corresponding to the HBV-S gene and the HCV-5' non-coding region were introduced into distinct plasmids. HBV plasmid was used as a standard for HBV-DNA quantitation, in competition with the HCV plasmid as internal control. HBV and HCV plasmids also served as template for transcription of HBV-RNA, and HCV-RNA, which was used as internal control and standard, respectively, in competition for HCV-RNA quantitation. The analyzed samples for HBV and HCV quantitation were processed in the same way in competition with the internal controls and to the respective calibration curves obtained by serial dilutions of the mimic standard. This method showed very good specificity and sensitivity, allowing absolute quantitation in a large linear range from 5 viral genomic copies per assay up to 10(6) copies, in sera of chronically HBV and HCV infected patients, as well as in supernatants of cell cultures inoculated with these viruses.

Interest of immunomodulation as a mean to improve the preparation of polyclonal and monoclonal antibody reagents.

Immunomodulation by monoclonal antibodies (mAbs) was investigated in mice in order to improve the preparation of antibody reagents. Three different types of representative immunogens were chosen: a human soluble protein (secretory immunoglobulin A, SIgA), a bacterial polysaccharide from E. coli K1 and an envelope protein from the hepatitis B virus. These Ag are all of importance for diagnosis and exhibit different levels of immunogenicity. Antibody-mediated enhancement was observed against restricted and defined regions of each immunogen i.e.: the Fab epitopes of SIgA, the preS1 domain of the HBV envelope and associated cell wall components of the capsular PS. The epitopes which were enhanced appeared to be different from those recognized by the modulating mAb. Negative modulations were also observed. Moreover, new epitopes seemed to be generated. In both cases the level and direction of the modulation were irrespective of isotypy and affinity of the mAbs. Interestingly the positive modulatory effect was found to be correlated with an in vitro assay based on the binding of immune complex to antigen-presenting cells.

[Incidence of sporadic hepatitis E in Ivory Coast based on still problematic serology]. / Fréquence de l'hépatite sporadique E en Côte d'Ivoire, vue au travers d'une sérologie encore problématique.

The first well-documented outbreak of viral hepatitis E in Africa was described in 1986 in Côte d'lvoire. Subsequently, no other outbreaks have been observed in the country. Côte d'lvoire therefore offers an excellent opportunity to evaluate the prevalence of sporadic viral hepatitis E in a country where the frequency of non-A, non-B, non-C viral hepatitis appears to be high. The study was carried out in Abidjan, the most populous city, and involved 111 hospitalized patients suffering from non-A, non-B and presumed non-C acute viral hepatitis. Screening for leptospirosis or a toxic etiology was carried out and the risk of including such patients eliminated. Diagnosis of viral hepatitis A was excluded from the absence of IgM anti-HAV antibodies. Patients with HBsAg and anti-HCV antibodies were not included in the study, although co-infection in asymptomatic HBV carriers or subsequent infection in patients who had recovered from a past HCV infection remained possible. There was a risk that some patients with late appearance of anti-HCV antibodies were included since PCR tests could not be performed. Cytomegalovirus or Epstein-Barr virus was not involved, since no specific IgMs against these viruses were detectable. Large discrepancies between the two commercial enzyme-linked immunosorbent assays (ELISAs) available for serological diagnosis of hepatitis E (Abbott and Genelabs) were observed. Among the 53 sera screened using both tests, only 20 gave positive results in both, and all such sera were confirmed using a domestic immunological test involving inhibition of labelled, well-documented anti-HEV-specific human IgG. Immunological confirmation was obtained for only half of the sera with discordant results in the commercial ELISAs. Full agreement between both commercial tests was observed for only 59% of the sera studied. The minimal incidence of sporadic viral hepatitis E among hospitalized patients in Abidjan with an acute hepatitis was estimated to be 27%.