Mario Perotti

Molecular diagnosis of sepsis in neutropenic patients with haematological malignancies.

The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMérieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.

Identification of a Broadly Cross-Reacting and Neutralizing Human Monoclonal Antibody Directed against the Hepatitis C Virus E2 Protein

ABSTRACT Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.

Hepatitis C Virus (HCV) Infection May Elicit Neutralizing Antibodies Targeting Epitopes Conserved in All Viral Genotypes

Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.

Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia

Hepatitis C virus (HCV) infection has been closely related to mixed cryoglobulinemia (MC). During HCV infection, cryoglobulins derive from the restricted expression of few germline genes as VH1-69, a subfamily highly represented in anti-HCV humoral response. Little is known about the self-reacting IgM component of the cryoprecipitate. In the present study, the IgM/K repertoire of an HCV-infected cryoglobulinemic patient was dissected by phage-display on well-characterized anti-HCV/E2 VH1-69-derived monoclonal IgG1/Kappa Fab fragments cloned from the same patient. All selected IgM clones were shown to react with the anti-HCV/E2 antibodies belonging to VH1-69 subfamily. More than 60% of selected clones showed a bias in VH gene usage, restricted to two VH subfamilies frequently described in autoimmune manifestations (VH3-23; VH3-21). Moreover, all selected clones showed an high similarity (>98.5%) to germline genes evidencing their natural origin. A possible hypothesis is that clones belonging to some subfamilies are naturally prone to react against other VH gene subfamilies, as VH 1-69. An antigen-driven stimulation of these subfamilies, and their overexpression as in HCV infection, could lead to a breaking of humoral homeostatic balance exposing the patients to the risk of developing autoimmune disorders.

Perspectives for the utilization of neutralizing human monoclonal antibodies as anti-HCV drugs☆

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

Antigen-Driven Evolution of B Lymphocytes in Coronary Atherosclerotic Plaques

Recent data indicated that adaptive immunity is involved in the process of atherogenesis. Oligoclonal recruitment of T lymphocytes has been described in coronary plaques of patients with acute coronary syndrome. However, the nature of immune response remains to be determined. In the present study, we examined the Ab response in six coronary plaques obtained by endoluminal directional atherectomy. The IgG1/κ-coding gene repertoires of B lymphocytes present in circulating blood and in coronary plaques were cloned and analyzed. In all of the six plaques, we observed 1) a skewed usage of heavy and light IgG1/κ Ab-coding genes, 2) an oligoclonal distribution of VK, JK, and VH, DH, and JH genes with overrepresentation of some rarely used IgG genes, and 3) the unequivocal signs of Ag-driven clonal expansion and evolution of B cells. The data document for the first time the presence of a local Ag-driven clonal evolution of B cells in human atherosclerotic plaques.

Molecular mycological diagnosis and correct antimycotic treatments.

In a recent report, Schaenman and colleagues (5) describe a case of a Scedosporium apiospermum soft tissue infection in an immunocompromised patient successfully treated with voriconazole. The article focuses on one of the hottest topics in current medical mycology, the emergence of antimycotic-resistant fungal isolates (3). Indeed, Scedosporium apiospermum is, also in our direct experience, one of the emerging fungal pathogens frequently endowed with resistance against drugs used as first-line agents (i.e., amphotericin B and fluconazole) (2). Therefore, we agree to the general message of the paper regarding the need of a prompt and well designed antifungal therapy. However, we would like to address a major point on how this could be achieved. In fact, we disagree that presumptive fungal identification based on aspecific morphological aspects is sufficient to take into account a new drug such as voriconazole as a first-line agent in the management of fungal infections. As admitted by the authors and clearly shown in Fig. 2 of their case report, many fungal genera feature morphological characteristics difficult to discriminate and the identification is not straightforward, especially if specific structures are not usually evident, as may be the case upon direct examination of clinical samples. A clinician making the same assumption as the authors might feel free to treat critically ill patients with voriconazole in the majority of cases, thus putting the whole community at risk for the emergence of new resistances to this valuable drug, as has already and inevitably happened for narrow-spectrum triazoles. Moreover it is still far from being proven that the toxicity profile of the new extended-spectrum triazoles is really safer than that of narrow-spectrum drugs, with severe side effects reported in up to 10% of patients receiving voriconazole (1). We think that a rapid and precise identification, at least at the genus level, is crucial for the prescription of a well designed empirical therapy, but we are convinced that it should be based on objective data. Recently, we addressed the diagnosis of mycotic keratitis using, in parallel with cultural methodologies, a molecular approach based on direct amplification from the biological sample and sequencing, by means of universal fungal primers (4, 6), of genus- and species-specific targets on the fungal genome. In our opinion this molecular approach allowing unequivocal identification of a fungal pathogen, at least at the genus level, in only one day is, together with a more thorough understanding of mechanisms of drug resistance, a real improvement of the conventional mycological diagnosis and represents a correct answer to the clinical questions posed by the availability of multiple classes of antifungal agents.