Stefania Manzara

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.

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.

Molecular Identification and Typing of Enteroviruses Isolated from Clinical Specimens

ABSTRACT Enterovirus characterization and typing require an integrated technological approach, using both immunological and molecular methods. The seventy-nine enteroviruses included in this study were isolated from cell cultures and classified as enteroviruses on the basis of an indirect immunofluorescence assay (IFA) against common enterovirus antigens and a neutralization test based on the Lim Benyesh-Melnick (LBM) pool. The final identification was carried out using a number of different molecular approaches, including reverse transcription (RT)-PCR, restriction fragment length polymorphism (RFLP) analysis, and nucleotide sequence analysis of amplicons from various regions of the genome. Twenty-seven poliovirus strains (set A) were identified using LBM pool analysis, RFLP analysis, and IFA. Use of the LBM pool method showed that 35 out of 79 strains were nonpoliovirus (set B), while 17 specimens tested negative (set C). Sets B and C were further investigated. Twenty-five specimens from set B and 8 from set C were identified by IFA. Six specimens from set B and five from set C were identified by RFLP analysis. Specimens in sets B and C were treated using RT-PCR; the resulting amplicons were subjected to nucleotide sequence analysis. The VP1 region was analyzed using two sets of deoxyinosine degenerate primers. Where the VP1 test gave no signal, the VP4-VP2 region was analyzed. Where both tests were negative, a 5′ noncoding region analysis was performed. Interestingly, analysis of the VP1 region showed that two specimens from set C were strains of enterovirus 71, whose presence was unexpected in Italy. As in other European epidemiological studies, the strain found most frequently was echovirus 30.

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.

Prospective evaluation of epstein-barr virus reactivation after stem cell transplantation: association with monoclonal gammopathy.

Epstein–Barr Virus (EBV) reactivation and EBV-related post-transplant lymphoproliferative disease (PTLD) have emerged as a severe complication after stem cell transplantation (SCT). We prospectively evaluated 104 consecutive patients receiving SCT either autologous or allogeneic. Fifty-two patients (50%) presented EBV DNA-emia and five of them developed PTLD proven or probable. PTLD rate was 9.6% among patients with EBV DNA-emia. One patient developed PTLD without EBV DNA-emia (0.96%). Overall PTLD incidence was 5.7%. No PTLD developed after autologous SCT. EBV DNA-emia was significantly more frequent after allogeneic than autologous SCT (60.7% vs 17.4%, p = 0.0002). At EBV reactivation, serum protein electrophoresis and immunofixation were assessed. Global incidence of γ-peak after allogeneic SCT with EBV reactivation was 65.3% (32/49 patients) and monoclonal gammopathy (MG) was identified in 23/28 evaluable patients (82%). All patients with PTLD developed γ-peak and in five of them MG was identified. MG is consistently associated with EBV DNA-emia and may help identification of progression to PTLD after allogeneic SCT.

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.

Prospective evaluation of epidemiological, clinical, and microbiological features of pandemic influenza A (H1N1) virus infection in Italy

Since several characteristics of pandemic influenza A (H1N1) virus infection remain to be determined, this study aimed to describe clinical features and complications of patients infected with H1N1. Subjects affected by influenza‐like illnesses and a control group of asymptomatic patients were enrolled prospectively at an Emergency Department from October 2009 to April 2010. At enrollment, clinical data and nasopharyngeal swabs for virological analyses were obtained. Ill subjects were followed until recovery and swabs were collected weekly in patients infected with H1N1. Of 318 patients enrolled, 92 (28.9%) were positive to H1N1. Patients infected with H1N1 were mainly young adults and complained classic influenza‐like symptoms. Fever was observed for a median time of 5 (IQR 3–7) days. Hospitalization occurred in 27.7% with 2% requiring intensive care unit admission: median length of hospitalization was 6 days (IQR 5–9). Pneumonia was diagnosed in 19.6% of patients. A similar proportion of lower airways involvement and of clinical complications was observed in subjects testing positive or negative for H1N1. However, patients infected with H1N1 were younger and hospitalized for a shorter period as compared to the control group (P = 0.002 and P = 0.045, respectively). Older age, asthma/chronic obstructive pulmonary disease and hypertension were associated with an increased risk of pneumonia. Viral shedding was observed for at least 1 week in 21.3% of patients. Asymptomatic infection was uncommon (1.1%). Respiratory syndromes caused by H1N1 and factors associated with disease severity were investigated and compared to influenza‐like illnesses of other origin. Such findings might contribute to improve clinical and epidemiological management of the disease. J. Med. Virol. 83:2057–2065, 2011.

Human Monoclonal Antibody Fragment Specific for Glycoprotein G in Herpes Simplex Virus Type 2 with Applications for Serotype-Specific Diagnosis

ABSTRACT A combinatorial library was used to select a human monoclonal antibody fragment (Fab) with high affinity for G glycoprotein in herpes simplex virus type 2 (HSV-2). Tests with 112 clinical specimens demonstrated successful discrimination between HSV-2 and HSV-1, showing the potential of Fab as a low-cost tool for HSV subtyping in clinical diagnosis.

Genomic studies on killer yeasts belonging to the genus Pichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida 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 theEcoRI andXhoI RFLP patterns withP. 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 andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.