Molecular characterization of adenovirus from clinical samples through analysis of the hexon and fiber genes.

Human adenoviruses (HAdVs) are common pathogens associated with a variety of clinical manifestations. Although most infections are self-limiting, HAdVs can cause severe or lethal infections in immunocompromised as well as in healthy individuals. Several HAdVs have recently been characterized as emerging pathogens. In Italy, epidemiological, and especially molecular epidemiological, information on this pathogen is scarce. This study describes the characterization by cell culture, PCR and phylogenetic analysis of HAdV strains originating from a small collection of clinical samples gathered between 2008 and 2009. The distribution of different HAdV species was studied and the possible presence of newly emerging types was ascertained. A broad-range primer pair was used, targeting a portion of the hexon gene, in combination with species-specific primer pairs targeting a portion of the fiber gene. Human and animal reference AdV strains were included in the study. The broad-range assay identified all HAdV strains (study and reference samples), as well as three out of four animal AdV reference strains. Seven different types belonging to three HAdV species (B, C and F) were identified in the study samples. Species C was by far the most frequent. Two co-infections were detected, each with two serotypes within species C (types 1/2 and 2/6). The combined use of these two PCR assays--allowing not only the identification of known types but also, potentially, the discovery of newly emerging ones--can provide valuable epidemiological information on the spread of HAdVs.

Clinical presentation, microbiological features and correlates of disease severity of 2009 pandemic influenza A (H1N1) infection.

The purpose of this study was to describe epidemiological, clinical and microbiological characteristics of confirmed novel influenza A (H1N1) infection, investigating factors associated with disease severity. We retrospectively selected patients seeking care for respiratory symptoms in two periods (May-August and September-November 2009) with different epidemiological characteristics. Only patients with confirmed pandemic influenza A (H1N1) were enrolled in this study. A total of 104 patients with H1N1 infection were evaluated, mostly referring classic influenza symptoms; in addition, diarrhea and vomiting were often referred. Clinical signs, symptoms and respiratory complications were different in the two periods. Of all patients, 18 (17%) had pneumonia. Patients older than 50 years showed a lower probability of pneumonia diagnosis when compared to children aged 0-13 (p = 0.049); a longer duration of symptoms before medical care was associated with a higher probability of pneumonia (p = 0.026). Phylogenetic analysis showed a low variability both in hemagglutinin and neuraminidase genes. In addition, no neuraminidase mutation associated with antiviral resistance was detected. A detailed description of respiratory diseases associated with H1N1 infection was provided and factors associated with its severity were investigated, thus contributing to the insight into epidemiological, clinical and microbiological knowledge of the disease.

A molecular method for the recovery and identification of enteric virus in shellfish.

In this paper we report the results of an investigation into the presence of enteric viruses in shellfish from the waters around Sardinia. Twenty two samples of shellfish were examined using a rapid and sensitive technique to concentrate and detect viral RNA in shellfish tissues. After recovery of viral particles, RNA was extracted, transcribed into cDNA and amplified using "nested PCR". Testing with enterovirus-specific RT-PCR produced positive results in over 13% of specimens. The virus detection procedure appears to be effective. In some circumstances it could be a better test of water quality than conventional monitoring techniques.

Cloning and characterization of human recombinant antibody Fab fragments specific for types 1 and 2 herpes simplex virus.

Twenty-six bacterial clones producing human recombinant Fab fragments specific for Herpes Simplex virus (HSV) antigens were obtained from an IgG1k human antibody combinatorial library displayed on filamentous phage, following panning against an HSV lysate. All the Fabs reacted against the HSV lysate in enzyme-linked immunosorbent assay and were able to recognize both type 1 and type 2 HSV in an indirect immunofluorescence assay (IFA). DNA sequencing of the heavy chain variable regions showed that these Fabs were different from those already described. One of these Fabs (Fab19) was purified and subjected to further characterization. Purified Fab19 was able to specifically recognize several different HSV-1 and HSV-2 strains (including 2 reference strains and 12 clinical isolates) in IFA. It was also able to neutralize the infectivity of both HSV-1 and HSV-2 strains, although the neutralizing activity was somewhat lower against HSV-2. In fact, 100% neutralization of infectivity was observed at a Fab concentration of 2 micrograms/100 TCD50 for the majority of HSV-1 strains, while a concentration of 8 micrograms/100 TCD50 was needed for 100% neutralization of all the HSV-2 strains tested. Owing to the above properties, Fab19 appears to be useful for diagnostic purposes and might also prove useful for in vivo immunoprophylaxis and therapy of HSV infections.

Genomic studies on killer yeasts belonging to the genus Pichia.

Twenty-four species belonging to the genus Pichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA. Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii and Candida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of the EcoRI and XhoI RFLP patterns with P. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis and C. albicans). However, one hybridized band, located at 3.9-4.1 Kb, seems to be peculiar to the Pichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.

In vitro susceptibility of 119 yeast isolates to fluconazole, 5-fluorocytosine, amphotericin B and ketoconazole.

The in vitro activity of fluconazole was tested against 13 yeast species (119 strains) isolated from clinical specimens during a 3-month period. For comparative purposes, three other antifungal compounds (5-fluorocytosine, amphotericin B and ketoconazole) were also tested. The tests were carried out using a microautomated method previously developed in our laboratory. The method allowed us to determine the minimum inhibitory concentration (MIC) of the four antifungal drugs used. For each of the drugs we utilized different media. The MIC ranges (mg/l) of fluconazole were: 0.04-12.5 for Candida albicans, 0.19-6.25 for Candida parapsilosis, 12.5-50 for Candida krusei, 0.04-100 for Candida tropicalis, 0.04- greater than 100 for Candida glabrata, 0.09- greater than 25 for Cryptococcus neoformans, 0.09-0.78 for Saccharomyces cerevisiae, 6.25- greater than 100 for Trichosporon beigelii and 0.09-0.19 for Blastoschizomyces capitatus (Trichosporon capitatum). The MIC value (mg/l) was 0.39 for Candida guilliermondii and Candida lusitaniae, greater than 100 for Cryptococcus laurentii and 0.09 for the 3 isolates of Torulopsis candida. These results were obtained using the medium recommended for in vitro testing of fluconazole (high-resolution medium) by Pfizer UK.

Epidemiological and clinical aspects of mycoses in patients with AIDS-related pathologies.

Mycological, cultural and/or serological studies were performed on 98 patients hospitalized in the Department of Infectious Diseases of the Catholic University in Rome with diagnoses of acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) diseases. The incidence of mycoses was evaluated by retrospectively analyzing the results of mycological examinations and comparing them with clinical manifestations. The presence of concomitant bacterial, viral and parasitic infections was also examined. For epidemiological purposes, the study was extended to include the biotyping of all yeasts isolated from patients hospitalized between September 1988 and February 1989 in the same Department. Antimycotic susceptibility was also determined for the first yeast isolate obtained from each of these patients. Oral candidiasis (50 cases) caused by Candida albicans was the most frequent mycosis, followed by esophageal candidiasis (13 cases) and cryptococcosis (6 cases). Four out of the 6 cryptococcosis patients had meningeal involvement. Systemic candidiasis (2 cases) and aspergillosis (1 case) were less common. Biotyping of yeasts isolated between September 1988 and February 1989 with the killer system revealed type 377 to be the most common among the C. albicans isolates. It represented 70% of all the yeasts isolated.

Serological study of yeast killer toxins by monoclonal antibodies.

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.

Biotyping of bacterial isolates using the yeast killer system.

Forty-four presumptive killer yeasts were tested against bacterial isolates, including rapid-growing gram-positive and gram-negative bacteria, as well as slow-growing bacteria, such as the mycobacteria. A killer system, based on the patterns of bacterial susceptibility to the action of nine selected killer yeasts, was developed for epidemiological purposes. The killer system, previously standardized for yeasts and hyphomycetes, was adapted to the specific growth conditions of the bacterial isolates. The results obtained confirm that susceptibility to the yeast killer phenomenon is widespread among microorganisms unrelated to yeasts and that it could form the basis for a convenient and adaptable biotyping method in microbiological laboratories.

Biotyping of aerobic actinomycetes by modified killer system.

Forty-four yeasts belonging to the genera Pichia, Candida, Saccharomyces and Kluyveromyces were tested for their potential killer effect on 13 aerobic actinomycetes (6 Nocardia asteroides, 1 N. brasiliensis, 1 N. caviae and 5 Actinomadura madurae). Only a few yeast strains did not display any killer activity against the aerobic actinomycetes studied, thus confirming that the killer phenomenon is widespread among microorganisms. For epidemiological purposes, a killer system was developed. According to their susceptibility to the 9 killer yeasts chosen, it was possible to differentiate the Nocardia and Actinomadura isolates into biotypes. Fitting conditions of the killer system to potential sensitive microorganisms with different characteristics of growth are also discussed.

Antibacterial activity of sisomicin.

A study of the in vitro antibacterial activity of a new aminoglycoside, sisomicin, is reported. The results show no significant differences between the activity of sisomicin and gentamicin.