Development and validation of a molecular method for the diagnosis of medically important fungal infections

The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.     

Candida albicans in a neonatal intensive care unit: antifungal susceptibility and genotypic analysis

Invasive candidiasis in neonates has become an increasing problem over the past decade in Neonatal Intensive Care Units (NICUs). From August 2005 to January 2006, six invasive candidiasis occurred in neonates in NICU of the S. Matteo hospital of Pavia. The study focused on the species involved and their in vitro antifungal susceptibility. Genotyping was conducted to determine clonal relatedness. A total of 22 yeasts were isolated from different biological samples of neonates during six months. The infants were infected with or colonized by Candida albicans and six patients developed C. albicans deep infections. The genotyping of the transposable intron region of C. albicans strains showed that they belonged to the genotype A (17 isolates) and genotype B (5 isolates). The RAPD confirmed these results. These data suggest that nosocomial transmission of C. albicans could be take into account as a mode of acquisition by neonates in NICUs at this hospital.     

Description of a fertilization-independent obligate apomictic species: Corunastylis apostasioides Fitzg

The Australian midge orchid Corunastylis apostasioides of the tribe Diurideae has completely eliminated any male contribution in the process of seed formation, which occurs directly from the maternal tissue by a process termed apomixis. Here, we report C. apostasioides to be an obligate apomictic species devoid of any sexuality and compare its development to a close sexual relative C. fimbriata (R. Br.) D.L. Jones & M.A. Clem. Apomictic characteristics in C. apostasioides include production of seed in absence of fertilization, frequently closed flowers, production of immature pollen in non-dehiscent anthers, expansion of ovaries despite the lack of fertilization and the absence of a citronella scent that is found in C. fimbriata produced to attract pollinating vinegar flies (Jones 2006). The nature of apomixis in C. apostasioides was examined by ovule histology and amplified fragment length polymorphism (AFLP) in each case drawing comparison with sexual C. fimbriata. In C. apostasioides the central megaspore mother cell undergoes diplosporic apomixis, while additional embryos are derived from nucellar or integument initials formed by sporophytic apomixis. Typical of apomicts, C. apostasioides is polyploid compared to the sexual C. fimbriata. The divergences of C. apostasioides from sexuality to apomictic development are discussed.     

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.     

Lymphocyte-facilitated infection of epithelia by human T-cell lymphotropic virus type I.

After the addition of human T-cell lymphotropic virus type I (HTLV-I)-infected lymphocytes to enterocyte monolayers, the lymphocytes adhered via microvilli from both cell types and shed virus onto the enterocyte surface. Virus fused with the epithelial membrane and infected these cells as confirmed by electron microscopic immunocytochemistry, in situ hybridization, and amplification by polymerase chain reaction.     

Activity of ovarian nitric oxide synthase (NOs) during ovulatory process in the rat: relationship with prostaglandins (PGs) production.

Nitric oxide (NO) is synthesized by the rat ovary and a role in the follicular development, the ovulation, and the luteal formation has been postulated. The aims this study were to determine the activity of nitric oxide synthase (NOs) enzyme during the ovulatory process and to demonstrate the existence of a relationship between the ovarian NO production and the synthesis of prostaglandins (PGs) involved in the follicular rupture. Prepuberal rats treated with PMSG/hCG to induce ovulation were used. The NOs activity, measured by [(14)C]citrulline formation, showed an increase after PMSG administration and reached a maximum at 10 h after hCG injection. NOs activity remained high up to 24 h post ovulation. At 10 h after the hCG injection, the activity of Ca(2+)-dependent NOs (constitutive NOs) was similar to that seen at 0 h, and the activity of Ca(2+)-independent NOs (inducible NOs) increased from 14.4 to 51% of total activity. The in vitro ovarian production of PGE and PGF(2alpha) was inhibited by L-NAME and stimulated by 3-morpho-linosydnonimine (SIN-1), a NO donor. The in vivo production of ovarian prostaglandins was also inhibited by the intrabursal administration of two NOs inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA). Our results suggest that the inducible NOs (iNOs) is the main isoform involved in the ovulatory process and that the NO produced stimulates the synthesis of both PGE and PGF(2alpha) from the cyclooxygenase pathway, to enhance the process of follicle rupture.     

Humoral immune response against hepatitis C virus

Antibodies are in several instances a reliable marker indicating vigorous immune response against infectious agents and in several viral diseases presence in the blood of specific anti-viral antibodies indicates an effective protection. However, this is not always true. For example, in the case of hepatitis C virus (HCV) an important human pathogen considered the causative agent of the nonA- nonB hepatitis, in spite of an intense antibody response there is no protection against a new infection and in the majority of infected individuals the virus overcomes host defences establishing a persistent infection. Here we describe how the dissection of the humoral immune response against HCV glycoprotein E2 of infected patients was useful for a better comprehension of the virus-host interplay. Cross-reactive antibodies directed against E2 are produced by the HCV-infected patient, but not all of them are protective, and some could even result to be detrimental for the patient. The cross-reactive anti-HCV/E2 humoral antibody response is complex and not necessarily completely beneficial to the host.     

Carrageenan formulation prevents macrophage trafficking from vagina: implications for microbicide development

Considerable evidence suggests that human immunodeficiency virus (HIV)-infected macrophages and/or lymphocytes may mediate sexual transmission of HIV. We and others have previously demonstrated that when vitally stained donor mouse lymphocytes or macrophages are placed in the vaginas of mice, some of the stained cells can later be found in the iliac lymph nodes. The aim of this study was to assess the extent of mononuclear cell trafficking from the vagina and to test the possibility that carrageenan formulation, a sulfated polysaccharide formulation containing 3% PDR98-15 carrageenan (PC-515; FMC Biopolymer, Rockland, ME), a vaginal microbicide, would prevent vaginal transmigration of macrophages. When supravitally stained mouse macrophages and T cells were inoculated into the vagina of recipient mice, discrete numbers of donor cells migrated to the recipient iliac and inguinal lymph nodes and spleen. When recipient mice were preinoculated with the carrageenan formulation, the number of macrophages in lymph nodes and spleen was reduced by >90%. In contrast, a methylcellulose formulation, which is believed to be inactive, did not significantly reduce migration to the lymphoid organs. Our findings suggest that the carrageenan formulation blocks cell trafficking of macrophages from vagina and that blocking does not result from cytotoxicity. Blocking cell trafficking may help to prevent sexual transmission of HIV.     

A very rare association of three mutations of the HFE gene for hemochromatosis

In the present paper, we describe a patient who is a compound heterozygote for three mutations in the HFE gene: C282Y, H63D, and E168Q. The patient's mother carries two copies of H63D and one copy of E168Q; the patient's father is heterozygous for C282Y. The family study indicates that the patient, as well as his sister, a maternal uncle, and a first cousin, all have inherited a single HFE allele that contains two mutations H63D and E168Q. The clinical symptoms and laboratory findings of the patient and his relatives are consistent with the conclusion that the E168Q mutation by itself is unlikely to result in hemochromatosis.