Bruno Pozzetto

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses.

Abstract Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1–4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.

High Sensitivity and Specificity of Serum Procalcitonin Levels in Adults with Bacterial Meningitis

It was shown in children that serum procalcitonin was the best marker to use to differentiate bacterial from viral meningitis. To evaluate procalcitonin in the diagnosis of acute bacterial and viral meningitis, we conducted a prospective study including adult patients who were suspected of having meningitis and who were admitted to an emergency department. Cerebrospinal fluid (CSF) and serum levels of procalcitonin were measured in 105 consecutive patients. The diagnosis of meningitis was based on clinical findings, gram staining, culture, and chemical analysis of CSF. Twenty-three patients had bacterial meningitis, 57 had viral meningitis, and 25 did not have meningitis. Bacteriologic and chemical analysis of CSF did not allow correct differentiation of viral from bacterial meningitis. On the other hand, a serum procalcitonin level >0.2 ng/mL had a sensitivity and specificity of up to 100% in the diagnosis of bacterial meningitis. Serum procalcitonin levels seem to be the best marker in differentiating between bacterial and viral meningitis in adults.

Enteroviral Capsid Protein VP1 Is Present in Myocardial Tissues From Some Patients With Myocarditis or Dilated Cardiomyopathy

BACKGROUND There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. METHODS AND RESULTS Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. CONCLUSIONS Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Toll‐like receptor 4 ligand can differentially modulate the release of cytokines by human platelets

Blood platelets link the processes of haemostasis and inflammation. This study examined the immunomodulatory factors released by platelets after Toll‐Like Receptor 4 (TLR4) engagement on their surfaces. Monoclonal anti‐human FcγRII Ab (IV.3)‐treated human platelets were cultured with TLR4 ligands in the presence or absence of blocking monoclonal antibody to human TLR4. The release of sCD62p, epidermal growth factor (EGF), transforming growth factor β (TGFβ), interleukin (IL)‐8, platelet activating factor 4 (PAF4), platelet‐derived growth factor, α, β polypeptide (PDGF‐AB), Angiogenin, RANTES (regulated upon activation, normal T‐cell expressed, and presumably secreted) and sCD40L were measured by specific enzyme‐linked immunosorbent assay. TLR4 ligand [Escherichia coli lipopolysaccharide (LPS)] bound platelet TLR4, which differentially modulates the release of cytokines by platelets. It was noted that (i) sCD62p, IL‐8, EGF and TGFβ release were each independent of platelet activation after TLR4 engagement; (ii) RANTES, Angiogenin and PDGF‐AB concentration were weaker in platelet supernatant after TLR4 engagement; (iii) sCD40L and PAF4 are present in large concentration in the releaseate of platelets stimulated by TLR4 ligand. The effects of LPS from E. coli on the modulation of secretory factors were attenuated by preincubation of platelets with an anti‐TLR4 monoclonal antibody, consistent with the immunomodulation being specifically mediated by the TLR4 receptor. We propose that platelets adapt the subsequent responses, with polarized cytokine secretion, after TLR4 involvement.

Cytomegalovirus load in inflamed intestinal tissue is predictive of resistance to immunosuppressive therapy in ulcerative colitis.

OBJECTIVES:Previous studies have suggested an association between cytomegalovirus (CMV) infection and steroid-refractory inflammatory bowel disease. In this study, the use of CMV DNA load during acute flare-ups of ulcerative colitis (UC) to predict resistance to immunosuppressive therapy was evaluated in intestinal tissue.METHODS:Forty-two consecutive patients (sex ratio M/F: 0.9, mean age: 43.6 years) hospitalized for moderate to severe UC and treated with IV steroids were included prospectively. A colonoscopy was performed for each patient at inclusion; colonic biopsy samples of the pathological tissue, and if possible, of the healthy mucosa, were tested for histological analysis and determination of CMV DNA load by real-time polymerase chain reaction assay. Patients were treated as recommended by the current guidelines.RESULTS:Sixteen patients were found positive for CMV DNA in inflamed intestinal tissue but negative in endoscopically healthy tissue; all of these patients were positive for anti-CMV IgG, three exhibited CMV DNA in blood, and none was positive for intestinal CMV antigen by immunohistochemistry detection. In the 26 remaining patients, no stigmata of recent CMV infection were recorded by any technique. By multivariate analysis, the only factor associated with CMV DNA in inflammatory tissue was the resistance to steroids or to three lines of treatment (risk ratio: 4.7; 95% confidence interval: 1.2–22.5). A CMV DNA load above 250 copies/mg in tissue was predictive of resistance to three successive regimens (likelihood ratio+=4.33; area under the receiver-operating characteristic curve=0.85). Eight UC patients with CMV DNA in inflamed tissue and therapeutic failure received ganciclovir; a clinical remission was observed in seven cases, with a sustained response in five of them.CONCLUSIONS:The CMV DNA load determined in inflamed intestinal tissue predicts resistance to steroid treatment and to three drug regimens in UC. Initiation of an early antiviral treatment in these patients might delay the occurrence of resistance to current treatments.

Comparison of Hepatitis C Virus NS5b and 5′ Noncoding Gene Sequencing Methods in a Multicenter Study

ABSTRACT A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5′ noncoding (5′ NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5′ NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5′ NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

Use of Flow Cytometry To Monitor Legionella Viability

ABSTRACT Legionella viability was monitored during heat shock treatment at 70°C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.

Genotypic and Phenotypic Analysis of Type III Secretion System in a Cohort of Pseudomonas aeruginosa Bacteremia Isolates: Evidence for a Possible Association between O Serotypes and exo Genes

The type III secretion system (TTSS) of Pseudomonas aeruginosa was characterized genetically and phenotypically in 92 epidemiologically unrelated bacteremic strains. Four groups of strains (TTSS types) were defined according to the level of type III protein secretion and kinetics of cytotoxicity. Type 1 strains (n=26) were highly and rapidly cytotoxic and secreted ExoU, type 2 strains (n=48) exhibited slower cytotoxic rates and expressed ExoS but not ExoU, type 3 strains (n=14) were poorly cytotoxic, and type 4 strains (n=4) were not cytotoxic. Type 3 and 4 strains did not have detectable secretion phenotype; however, some type 4 strains were able to reach a level of cytotoxicity similar to that of type 1 and type 2 strains when complemented in trans by a functional exsA gene. A statistically significant association (P<.001) was found between TTSS types and detection of the mutually exclusive exoU and exoS genes. In addition, 24 of 25 serotype O:1, O:10, and O:11 strains contained exoU, whereas 54 of 55 serotype O:3, O:4, O:6, O:12, and O:16 strains contained exoS (P<.001). Our results demonstrate correlations among exoU or exoS genotype, TTSS phenotype, and O serotype in bacteremic P. aeruginosa isolates.

Platelets and infections - complex interactions with bacteria.

Platelets can be considered sentinels of vascular system due to their high number in the circulation and to the range of functional immunoreceptors they express. Platelets express a wide range of potential bacterial receptors, including complement receptors, FcγRII, Toll-like receptors but also integrins conventionally described in the hemostatic response, such as GPIIb–IIIa or GPIb. Bacteria bind these receptors either directly, or indirectly via fibrinogen, fibronectin, the first complement C1q, the von Willebrand Factor, etc. The fate of platelet-bound bacteria is questioned. Several studies reported the ability of activated platelets to internalize bacteria such as Staphylococcus aureus or Porphyromonas gingivalis, though there is no clue on what happens thereafter. Are they sheltered from the immune system in the cytoplasm of platelets or are they lysed? Indeed, while the presence of phagolysosome has not been demonstrated in platelets, they contain antimicrobial peptides that were shown to be efficient on S. aureus. Besides, the fact that bacteria can bind to platelets via receptors involved in hemostasis suggests that they may induce aggregation; this has indeed been described for Streptococcus sanguinis, S. epidermidis, or C. pneumoniae. On the other hand, platelets are able to display an inflammatory response to an infectious triggering. We, and others, have shown that platelet release soluble immunomodulatory factors upon stimulation by bacterial components. Moreover, interactions between bacteria and platelets are not limited to only these two partners. Indeed, platelets are also essential for the formation of neutrophil extracellular traps by neutrophils, resulting in bacterial clearance by trapping bacteria and concentrating antibacterial factors but in enhancing thrombosis. In conclusion, the platelet–bacteria interplay is a complex game; its fine analysis is complicated by the fact that the inflammatory component adds to the aggregation response.

Development and Evaluation of Chlamylege, a New Commercial Test Allowing Simultaneous Detection and Identification of Legionella, Chlamydophila pneumoniae, and Mycoplasma pneumoniae in Clinical Respiratory Specimens by Multiplex PCR

ABSTRACT This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 × 10−3 IFU, 5 × 10−2 color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.