M. A. De Francesco

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.

The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.

Phenotypes, genotypes, serotypes and molecular epidemiology of erythromycin-resistant Streptococcus agalactiae in Italy

The purpose of this investigation was to analyse Streptococcus agalactiae (group B Streptococcus, GBS) isolates collected in Italy from vaginal and urine samples in respect to their clonality, distribution of virulence factors and antimicrobial resistance determinants. Three hundred and eighty-eight GBS were recovered from clinical samples. They were analysed for antibiotic resistance profiling. Erythromycin-resistant strains were further characterised by multilocus sequence typing (MLST), serotyping and the detection of alp genes of the alpha-like protein (Alp) family. GBS isolates represented 40 different sequence types (STs), grouped in five clonal complexes (CCs) and belonged to seven serotypes. Most serotype V strains (81%) possessed alp2-3; serotype Ia carried mainly epsilon, while the serotype III mainly rib. All isolates were susceptible to penicillin, whereas resistance to erythromycin was detected in 15% of isolates. Most erythromycin-resistant GBS strains were of serotype V (56.8%) and belonged to the CC-1 group (50%). Macrolide resistance phenotypes were the cMLSB (46.5%) and the M phenotypes (46.5%) due to the presence of ermB and mefA/E genes, respectively. These results provide data which establish a baseline for monitoring erythromycin resistance in this region and also provide an insight into the correlation among clonal types, serotypes, surface protein and resistance genes. The increased prevalence of strains that displayed the M phenotype strengthens the importance of the epidemiological surveillance of macrolide resistance in GBS, which may also represent an important reservoir of resistance genes for other species.

Prevalence of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa in an Italian hospital

The severity and extent of disease caused by multidrug-resistant organisms (MDROs) varies by the population(s) affected and the institution(s) at which these organisms are found; therefore, preventing and controlling MDROs are extremely important. A retrospective study of patients who were infected with Acinetobacter baumannii or Pseudomonas aeruginosa was performed at the Spedali Civili Hospital in Brescia, Italy, from 2007 to 2010. A total of 167 (0.52%) A. baumannii isolates and 2797 P. aeruginosa (8.7%) isolates were identified among 31,850 isolates. Amikacin and colistin were the most active agents against A. baumannii strains. Multidrug resistance (MDR) was observed in 57 isolates (54%). Most MDR isolates (42 out of 57, 73%) were resistant to four classes of antibiotics. P. aeruginosa was recovered more frequently from the respiratory tract, followed by the skin/soft tissue, urine and blood. Colistin, amikacin and piperacillin/tazobactam were active against 100%, 86% and 75% of P. aeruginosa isolates, respectively. A total of 20% (n=316) of P. aeruginosa isolates were MDR. In summary, A. baumannii was more rare than P. aeruginosa but was more commonly MDR. Epidemiological data will help to implement better infection control strategies, and developing a local antibiogram database will improve the knowledge of antimicrobial resistance patterns in our region.

Detection and identification ofMycobacterium avium in the blood of AIDS patients by the polymerase chain reaction

One hundred fifty-three blood samples from patients positive for the human immunodeficiency virus (HIV) were analyzed by polymerase chain reaction (PCR) to detect the presence ofMycobacterium avium. Samples were collected from patients who also had blood cultures performed by a radiometric method. Blood samples were centrifuged on a Ficoll-Hypaque gradient to purify peripheral blood mononuclear cells. The purified cells were washed and incubated with a resin, boiled to release mycobacterial DNA, and then amplified. Polymerase chain reaction products were detected by a nonisotopic method. A 123 base-pair (bp) insertion sequence, namely IS6110, fromMycobacterium tuberculosis complex was also included in the reaction as an internal control ofTaq polymerase activity to exclude the presence of enzyme inhibitors. This IS6110 fragment can be distinguished from the 383 bp target product on ethidium bromide-stained agarose gel and may also be used in a colorimetric assay. Such results were compared with the results of culture and indicated that the assay is as sensitive as bacteriological methods, though faster.

IFN-gamma restores HIV- and non-HIV-specific cell mediated immune response in vitro and its activity is neutralized by antibodies from patients with AIDS.

The addition of IFN‐γ to cultures of peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic HIV‐infected patients increased cell proliferation in response to HIV envelope synthetic peptides (Env), influenza A virus (VIRUS), andallogeneic lymphocytes (ALLO) but not to phytohaemagglutinin (PHA) stimulation. F(Ab)2 fragments of IgG purified from the sera of HIV‐seropositive patients specifically interfered with IFN‐γ‐induced cell proliferation in response to recallantigens. Neutralization of the lymphokine activity was found to be sustained by specific IFN‐γ antibodies. Data obtained demonstrate that IFN‐γ can restore the cell‐mediated immunity of a number of asymptomatic HIV+ individuals invitro, while IFN‐γ antibodies present in sera of patients with AIDS interfere with the activity of the lymphokine.

Emergence of the first levofloxacin-resistant strains of Streptococcus agalactiae isolated in Italy

ABSTRACT Of 901 group B streptococcus strains analyzed, 13 (1.4%) were resistant to levofloxacin (MICs of >32 μg/ml for seven isolates, 2 μg/ml for four isolates, and 1.5 μg/ml for four isolates). Mutations in the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV were identified. A double mutation involving the Ser-81 change to Leu for gyrA and the Ser-79 change to Phe or to Tyr for parC was linked to a high level of fluoroquinolone resistance. In addition, two other mutational positions in parC were observed, resulting in an Asp-83-to-Tyr substitution and an Asp-83-to-Asn substitution. Different mutations were also observed in gyrB, with unknown significance. Most levofloxacin-resistant GBS strains were of serotype Ib and belonged to sequence type 19 (ST19) and clonal complex 19 (CC-19). Most of them exhibited the epsilon gene.

Purification of natural human IFN-γ antibodies

Abstract Natural antibodies to interferon gamma (IFN-γ) were found in patients suffering from various viral infections, but also at weak titers in healthy individuals. In the present study we describe a one-step chromatographic procedure for the purification of the anti-IFN-γ antibodies from human Ig preparations, using a recombinant IFN-γ-coupled Sepharose CL4B affinity column. The antibodies to IFN-γ were eluted from the column using 3 different methods without loss of immunological activity. They were found to be Ig, mostly of the IgG1 subclass, and, in the biological assay, to be able to neutralize the de novo expression of Fc receptor sites induced by IFN-γ on U937 cells.

Invasive Candidiasis in Brescia, Italy: Analysis of Species Distribution and Antifungal Susceptibilities During Seven Years

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a large teaching hospital in Brescia, Italy, and the in vitro antifungal susceptibility of isolates. We analyzed 196 isolates causing fungemia in patients admitted in our hospital, between January 2009 and December 2015. Strains were identified by VITEK 2 and MALDI-TOF MS. MICs were determined by Sensititre Yeast OneTM. The resistance was defined by using the revised CLSI breakpoints/epidemiological cutoff values to assign susceptibility or wild type to systemic antifungal agents. Most infections were caused by Candida albicans (60%), Candida parapsilosis (15%), Candida glabrata (12%) and Candida tropicalis (6%). The susceptibility rate for fluconazole was 96.5%. Non-Candida species isolates exhibited full susceptibilities to echinocandins according to CLSI breakpoints. Amphotericin B demonstrated excellent activity against all Candida species. Local epidemiological and antifungal susceptibility studies are necessary in order to improve empirical treatment guidelines.

A cluster of invasive listeriosis in Brescia, Italy.

Clinical forms were indicated as bacteraemia, miscarriage and peritonitis. Listeriosis was considered hospital-acquired when signs of infection occurred ≥72 h after admission. For each patient with listeriosis, different data were analyzed retrospectively using a medical chart review: demographic information, underlying diseases preceding the onset of listeriosis; days from admission to onset of listeriosis, type of infection and outcome, antibiotic used for therapy. Data on the type of food consumed could not be collected because of insufficient information in medical charts for both the period before admission and during the hospital stay. Before the organism was isolated from microbiological specimens, Listeria had not been considered as being the causative organism in any of these cases. Gram-positive, nonspore forming rods, facultative anaerobes, which produced catalase and a narrow hemolysis, were presumptively identified as Listeria. Further identification was attained using VITEK (bioMérieux, St. Louis, MO, USA) and real-time PCR (Eusepscreen, Eurospital SpA, Trieste, Italy). Genotyping was performed by a multiplex PCR that separates the four major serovars (1/2a, 1/2b, 1/2c, and 4b) of L. monocytogenes strains into four distinct groups as previously described [7]. DNA was extracted from each isolate by the NucliSENS easyMAG (Biomèrieux, Florence, Italy), according to the manufacturer’s instructions. Approximately 1 ng of DNA was used in the PCRs with primers and conditions as described elsewhere [7]. The PCR products were identified by analyzing the unique banding pattern following agarose gel 2 % (wt/vol) electrophoresis. An automated DiversiLab system (BioMérieux), which uses repetitive extragenic palindromic sequencebased PCR (rep-PCR), was performed to further characterize the Listeria isolates. Listeria monocytogenes is a gram-positive facultative intracellular organism that frequently contaminates foodprocessing environments. Increasing incidence rates of invasive listeriosis have been reported over the last few decades by several European countries, with an incidence of listeriosis ranging from 0.3 to 0.8/100,000 people/year in France and Italy, respectively [1]. L. monocytogenes causes an invasive disease with the involvement of central nervous system and bactaeremia with a high case fatality (20– 30 %). Various risk factors have been related to invasive listeriosis. Older adults, where there is an immunosenescence of T-cell-mediated immunity, patients receiving immunosuppressive therapies and people with impaired cell-mediated immunity or autoimmune diseases are at higher risk of invasive listeriosis. Maternal infections are often asymptomatic or mild, but pregnant women can transmit the infection to the fetus, for whom the infection can be fatal [2]. Listeriosis is mainly transmitted through the consumption of contaminated food in the community, and vertical transmission from mother to child. Hospital-associated infections usually occur as clustered outbreaks from environmental contact [3, 4] or contaminated food [5, 6]. Here, we describe a cluster of six cases of invasive listeriosis identified at the Spedali Civili Hospital in Brescia, Italy, between July 2013 and February 2014. A listeriosis case was defined as isolation of L. monocytogenes from a normally sterile site such as blood or cerebrospinal fluid (CSF) or from placental or fetal tissue.

Comparative Evaluation of BDProbeTec ET, LCx and PACE 2 Assays for the Detection of Chlamydia trachomatis in Urogenital Specimens

Chlamydia trachomatis is probably the most widespread sexually transmitted disease in Western industrialised countries, with a trail of dramatic consequences. The organism is notoriously difficult to detect, which accounts for its spread and makes it so unpredictable, but once detected, the infection can be treated easily. Chlamydial infections are usually asymptomatic, or they cause mild or nonspecific symptoms and signs that are not likely to be noticed. Approximately 70% of women with endocervical infections and up to 50% of men with urethral infections are asymptomatic and therefore unlikely to seek medical attention [1]. Thus, chlamydia has become known as the silent epidemic. It is the most frequently identified single cause of pelvic inflammatory disease, which affects an estimated 15–40% of women [2]. Yet, effective screening for this agent can lead to prompt treatment and prevent complications. Non-culture, non-amplification methods such as enzyme immunoassay and probe hybridization (PACE 2 and PACE 2 PCA assays; Gen-Probe, USA) are now the most widely used tests for Chlamydia trachomatis screening [3, 4, 5, 6]. New amplification assays are gaining wider use because they have proven to be more sensitive than nonamplification tests and cultures [5, 6, 7, 8]. Additionally, they offer the advantage that urine can be used as an alternative specimen for testing [6, 7]. The purpose of the study presented here was to evaluate two commercially available amplification methods, the ligase chain reaction (LCx assay; Abbott Laboratories, USA) and the strand displacement amplification method (BDProbeTec Et; [BDPT] Becton Dickinson, USA), and compare them with two standard probe hybridization assays (PACE 2 and PACE 2PCA) using female endocervical and male urethral swabs from a population at low risk of chlamydia infection. Also reported here are the results of a preliminary study based on our analysis of the diagnostic performance of the two commercial amplification assays using first-void urine samples from male and female patients. The study population included 300 patients (239 women and 61 men), aged 18–50 years (mean age, 24 years) treated at Spedali Civili Hospital in Brescia, Italy, between March and June 2001. Fifty subjects (37 women and 13 men) aged 28–50 years, suffered from probable infertility (defined as the inability to achieve pregnancy after normal unprotected intercourse over a 1-year period); these patients were asymptomatic and had received no antimicrobial therapy in the previous month. The remaining 202 women, aged 18–45 years, were seen for the following reasons: routine examination during pregnancy (113 patients), routine gynecologic care (56 patients), and pregnancy ending in premature delivery and abortion (33 patients). The remaining 48 men, aged 28–50 years, were seen for clarification of a variety of symptoms. Preliminary information including patient age, reason(s) for examination, patient complaints and clinical assessment, and antibiotic use during the previous 21 days was obtained from each patient by the hospital’s nursing staff. Three endocervical or urethral samples were collected from each of the 300 patients according to the gender of the patient (239 women and 61 men). The first swab was used for Gram staining and then for BDPT. The second swab was used for a DNA probe (PACE 2), and the third swab was used for the LCx assay. A subset of 50 patients (37 women and 13 men) also provided urine samples, which were assayed by both amplification methods (BPDT and LCx). All samples were transferred to the laboratory in the respective manufacturer’s transport medium and stored according to the respective manufacturer’s instructions prior to testing. All tests were performed according to each manufacturer’s directions. C. Pollara ()) · L. Terlenghi · M. A. De Francesco · F. Gargiulo · F. Perandin · N. Manca Institute of Microbiology and Virology, Spedali Civili Hospital, Piazza Spedali Civili 1, 25123 Brescia, Italy e-mail: pollara@bshosp.osp.unibs.it Tel.: +39-030-3995860 Fax: +39-030-3996071