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Purpose: The Epstein–Barr virus (EBV) is present in the malignant Hodgkin/Reed–Sternberg (HRS) cells of 20% to 40% cases of Hodgkin lymphoma (HL) in Western countries. We were interested in the detection and quantification of cell-free plasma EBV-DNA as an indicator of biological and clinical characteristics in EBV-associated HL.Experimental Design: EBV was detected in peripheral blood compartments (whole blood, plasma, and mononuclear cells) at diagnosis by real-time PCR for the EBNA (EB nuclear antigen) region (n = 93) and in HRS cells by in situ hybridization for EBV-encoded small RNAs (EBER; n = 63). These data were correlated to histological and clinical characteristics, EBV serology, circulating cell-free DNA, and interleukin (IL)-6 levels.Results: Detection of EBV-DNA in plasma had a high specificity (90%), but a relatively low sensitivity (65%) to predict for EBV association. The viral load was higher in patients with advanced stage disease, older age in the presence of B-symptoms, and international prognostic score more than 2. The presence of EBV in HRS cells and higher plasma EBV-DNA copy numbers correlated to an increased frequency of tumor-infiltrating CD68+ macrophages in lymph node biopsies. Plasma EBV-DNA load correlated to circulating cell-free DNA and IL-6 levels, and inversely correlated to lymphocyte counts and EBNA1 antibody titers.Conclusion: Although the presence of EBV-DNA in peripheral blood cannot be regarded as a surrogate marker for EBER, the plasma EBV-DNA load at HL diagnosis is an indicator of disease activity and biological characteristics associated with negative prognosis. Moreover, the inverse correlation to EBNA1 antibody titers and lymphocyte counts may indicate a reduction in immunosurveillance, favoring the expansion of EBV-HRS cells in HL. Clin Cancer Res; 17(9); 2885–92. ©2011 AACR.

Supplementary Figure S1.

Purpose In presence of indeterminate lesions by fine needle aspiration (FNA), thyroid cancer cannot always be easily diagnosed by conventional cytology. As a consequence, unnecessary removal of thyroid gland is performed in patients without cancer based on the lack of optimized diagnostic criteria. Aim of this study is identifying a molecular profile based on long noncoding RNAs (lncRNAs) expression capable to discriminate between benign and malignant nodules. Methods Patients were subjected to surgery (n = 19) for cytologic suspicious thyroid nodules or to FNA biopsy (n = 135) for thyroid nodules suspicious at ultrasound. Three thyroid-specific genes (TG, TPO, and NIS), six cancer-associated lncRNAs (MALAT1, NEAT1, HOTAIR, H19, PVT1, MEG3), and two housekeeping genes (GAPDH and P0) were analyzed using Droplet Digital PCR (ddPCR). Results Based on higher co-expression in malignant (n = 11) but not in benign (n = 8) nodules after surgery, MALAT1, PVT1 and HOTAIR were selected as putative cancer biomarkers to analyze 135 FNA samples. Cytological and histopathological data from a subset of FNA patients (n = 34) were used to define a predictive algorithm based on a Naïve Bayes classifier using co-expression of MALAT1, PVT1, HOTAIR, and cytological class. This classifier exhibited a significant separation capability between malignant and benign nodules (P Conclusions ddPCR analysis of selected lncRNAs in FNA biopsies appears a suitable molecular tool with the potential of improving diagnostic accuracy.

OBJECTIVES: Several studies have documented the potent in vitro activity of caspofungin against Candida spp. This is of special concern for Candida glabrata infections that are often resistant to many azole antifungal agents and, consequently, difficult to treat. The aim of the present study was to expand the data on the in vitro activity of caspofungin against azole-resistant isolates of C. glabrata. METHODS: A total of 50 clinical isolates of C. glabrata were tested for susceptibility to caspofungin. The isolates were cross-resistant to multiple azoles, including fluconazole, itraconazole, ketoconazole and voriconazole. Expression of the resistance-related CgCDR1 and CgCDR2 genes was evaluated by quantitative RT-PCR analysis. The MICs of caspofungin were determined by using the National Committee for Clinical Laboratory Standards M27-A2 reference method. RESULTS: C. glabrata isolates exhibited increased expression of the CDR efflux pump(s), and this was in accordance with their high-level azole resistance. In contrast, all the isolates were highly susceptible to caspofungin (100% of isolates were inhibited at

The authors show the results of an integrated model for risk management of tuberculosis in a sample of sheltered homeless in Rome. Tuberculin skin test (TST) was used for evaluating the prevalence of latent infection (LTBI). In TST positives, expectorate was collected and chest X-ray was achieved. Multiple logistic regression analysis was performed to investigate determinants of infection. Out of 288 recruited subjects, 259 returned for the TST reading; 45.56% were positive and referred to a specialized center; 70 accessed the health facility and completed the clinical pathway. The risk factors associated to LTBI were male gender (OR=3.72), age over 60 years (OR=3.59), immigrant status (OR=3.73), and obesity (OR=2.19). This approach, based on an integrated social network, guarantees high adherence to screening (89.93%), allowing patients testing positive for latent tuberculosis infection to be diagnosed and rapidly referred to a specialized center.

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

Culture-independent molecular techniques such as real-time PCRs offer the potential for early diagnosis of invasive aspergillosis (IA), thereby reducing the disease-associated mortality rate. PCR-based testing is presently excluded from disease-defining consensus criteria due to lack of standardization and clinical validation. A single-center prospective study was conducted to investigate the performance of the commercially available MycAssay Aspergillus test for detecting Aspergillus DNA in patients with suspicion of IA. To this end, a total of 158 bronchoalveolar lavage (BAL) fluid specimens that were consecutively collected from hematology ( n = 68) and intensive care unit ( n = 90) patients were examined. Sixteen of 17 (94.1%) specimens from patients with proven/probable IA were MycAssay positive, and 15 of these 16 patients were also positive by an “in-house” PCR assay. A total of 139 of 141 (98.6%) specimens from patients without proven/probable IA were MycAssay negative. Fifteen of 16 (94.1%) MycAssay-positive patients were also positive for BAL fluid galactomannan (GM) at an index cutoff of ≥1.0 (index range, 1.1 to 8.3), as were 3 patients without IA but with pulmonary fusariosis. Interestingly, in seven of the PCR-positive BAL specimens that tested culture positive for Aspergillus species, cycle threshold values were earlier than those of specimens with a culture-negative result. In conclusion, the MycAssay Aspergillus PCR appears to be a sensitive and specific molecular test for the diagnosis of IA, and its performance is comparable to that of the GM assay. However, more large studies are necessary to firmly establish its clinical utility in high-risk settings.

Septic pulmonary embolism (SPE) is an uncommon, but life-threatening event that is usually associated with extrapulmonary infections. We report the first case of bilateral SPE secondary to a central venous catheter-related bloodstream infection involving pathogens commonly considered environmental contaminants: Tsukamurella tyrosinosolvens and Rhizobium radiobacter. Empirical levofloxacin treatment was confirmed by in vitro susceptibility data and produced prompt clinical improvement, but removal of the infected line proved indispensable for eradication of the infection. Laboratory personnel should be aware of the pathogenic potential of these environmental organisms, particularly in immunocompromised hosts with indwelling catheters.

Purpose: The Epstein–Barr virus (EBV) is present in the malignant Hodgkin/Reed–Sternberg (HRS) cells of 20% to 40% cases of Hodgkin lymphoma (HL) in Western countries. We were interested in the detection and quantification of cell-free plasma EBV-DNA as an indicator of biological and clinical characteristics in EBV-associated HL. Experimental Design: EBV was detected in peripheral blood compartments (whole blood, plasma, and mononuclear cells) at diagnosis by real-time PCR for the EBNA (EB nuclear antigen) region (n = 93) and in HRS cells by in situ hybridization for EBV-encoded small RNAs (EBER; n = 63). These data were correlated to histological and clinical characteristics, EBV serology, circulating cell-free DNA, and interleukin (IL)-6 levels. Results: Detection of EBV-DNA in plasma had a high specificity (90%), but a relatively low sensitivity (65%) to predict for EBV association. The viral load was higher in patients with advanced stage disease, older age in the presence of B-symptoms, and international prognostic score more than 2. The presence of EBV in HRS cells and higher plasma EBV-DNA copy numbers correlated to an increased frequency of tumor-infiltrating CD68+ macrophages in lymph node biopsies. Plasma EBV-DNA load correlated to circulating cell-free DNA and IL-6 levels, and inversely correlated to lymphocyte counts and EBNA1 antibody titers. Conclusion: Although the presence of EBV-DNA in peripheral blood cannot be regarded as a surrogate marker for EBER, the plasma EBV-DNA load at HL diagnosis is an indicator of disease activity and biological characteristics associated with negative prognosis. Moreover, the inverse correlation to EBNA1 antibody titers and lymphocyte counts may indicate a reduction in immunosurveillance, favoring the expansion of EBV-HRS cells in HL. Clin Cancer Res; 17(9); 2885–92. ©2011 AACR.

Thirty consecutive Acinetobacter baumannii isolates producing carbapenem-hydrolysing oxacillinases, OXA-23 or OXA-58, were recovered from patients hospitalized in Rome, Italy, between January and November 2007. Among these isolates, two clones not associated with the European clones I or II were observed. The oxacillinase-encoding genes were plasmid- or chromosome-borne. This study reports the dissemination of carbapenem-resistant A. baumannii belonging to two clones among several units in a single hospital and emphasizes the ability of A. baumannii to cause epidemic/endemic outbreaks and also to acquire various resistance genes circulating in the hospital environment.

Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

Adenoviruses are emerging pathogens which may represent new indicators of microbial water quality. In the present study, environmental samples of seawater, estuarine water, and influents of sewage treatment plants underwent both standard bacteriological and viral analyses (adenovirus identification, typing and quantification) in order to evaluate the role of surface water contamination as a possible vehicle for the transmission of adenovirus, and the relevance of adenoviruses as an additional tool in water quality assessment. Qualitative PCR methods were used for the detection and typing of adenoviruses. This was done through the sequencing and phylogenetic analysis of segments of the hexon- and fiber-coding regions of the viral genome. Subsequently, quantitative PCR assays based on TaqMan probe hydrolysis technology were used to assess virus concentrations in environmental samples. Results showed a widespread presence of adenovirus in the environment, even in the absence of bacterial indicators, confirming the relevance of evaluating these viruses as possible indicators of viral contamination of water.

The intracellular survival in mouse peritoneal macrophages of 8 Enterococcus faecalis response regulator mutants was tested to assess if the corresponding 2-component signal transduction systems (TCS) are involved in the ability of E. faecalis to survive in macrophages. Three mutants (err04, err05 and err06) are more susceptible than the wild-type JH2-2 strain and 1 is more resistant (err10). Then, characterization of the TCS Err04-Ehk04 and Err06-Ehk06 reveals that the first (homolog of PhoP-PhoR of Bacillus subtilis) is induced in phosphate deprivation conditions, regulates its own expression and plays a role in the expression of pstF encoding a phosphate-binding protein. The Err06-Ehk06 is involved in oxidative stress response. A mutation in the err06 gene increases sensitivity of the bacterium to H2O2. The err06-ehk06 operon is induced by H2O2 stress and controlled by 2 transcriptional start sites, of which 1 is specifically active in oxidative stress conditions. We also demonstrated that the expression of the catalase gene (kat) is partly dependant of the Err06-Ehk06 TCS.

Nosocomial Candida bloodstream infections rank among infections with highest mortality rates. A retrospective cohort analysis was conducted at Catholic University Hospital to estimate the risk factors for mortality of patients with candidemia. We reviewed records for patients with a Candida bloodstream infection over a 5-year period (January 2000 through December 2004). Two hundred ninety-four patients (42.1% male; mean age ± standard deviation, 65 ± 12 years) were studied. Patients most commonly were admitted with a surgical diagnosis (162 patients [55.1%]), had a central venous catheter (213 [72.4%]), cancer (118 [40.1%]), or diabetes (58 [19.7%]). One hundred fifty-four (52.3%) patients died within 30 days. Of 294 patients, 168 (57.1%) were infected by Candida albicans , 64 (21.7%) by Candida parapsilosis , 28 (9.5%) by Candida tropicalis , and 26 (8.8%) by Candida glabrata . When fungal isolates were tested for biofilm formation capacity, biofilm production was most commonly observed for isolates of C. tropicalis (20 of 28 patients [71.4%]), followed by C. glabrata (6 of 26 [23.1%]), C. albicans (38 of 168 [22.6%]), and C. parapsilosis (14 of 64 [21.8%]). Multivariable analysis identified inadequate antifungal therapy (odds ratio [OR], 2.35; 95% confidence interval [95% CI], 1.09 to 5.10; P = 0.03), infection with overall biofilm-forming Candida species (OR, 2.33; 95% CI, 1.26 to 4.30; P = 0.007), and Acute Physiology and Chronic Health Evaluation III scores (OR, 1.03; 95% CI, 1.01 to 1.15; P < 0.001) as independent predictors of mortality. Notably, if mortality was analyzed according to the different biofilm-forming Candida species studied, only infections caused by C. albicans ( P < 0.001) and C. parapsilosis ( P = 0.003) correlated with increased mortality. Together with well-established factors, Candida biofilm production was therefore shown to be associated with greater mortality of patients with candidemia, probably by preventing complete organism eradication from the blood.

Objectives. To analyze virological and immunological features of AIDS-related progressive multifocal leukoencepalophathy (PML) and their association to disease prognosis. Methods. In HIV-infected patients with virologically confirmed PML, JC virus (JCV) DNA load and levels of Macrophage Chemoattractant Protein (MCP)-1 were determined in cerebrospinal fluid. JCV genotypes, rearrangements and JCV DNA binding sites for cellular transcription factors were analyzed by sequencing the viral VP1 region and regulatory region (RR). Results. 45 patients were analyzed: 60% were exposed to highly active antiretroviral therapy (HAART) after PML and 24% before the disease onset. JCV DNA load in cerebrospinal fluid was a strong predictor of patients survival. Lower levels of JCV DNA in cerebrospinal fluid were associated with the following virologic factors: viral genotype 4 (p = 0.043), more rearrangements in the RR (p = 0.046), duplication of RR block B (p = 0.028), and duplication of binding sites for cellular transcription factor NF-1 (p = 0.060). In patients with prior antiretroviral exposure there was a trend towards a higher number of binding sites for cellular transcription factors (p = 0.068). Lower JCV load was also predicted by exposure to HAART (p = 0.010), higher baseline CD4 counts (p = 0.009) and higher cerebrospinal fluid MCP-1 levels (p = 0.036). In a multiple regression model, MCP-1 levels were independently associated with JCV load. Conclusion. HAART leads to a partial immune-mediated control of JCV replication; the virus may tend to escape through the selection of rearrangements in the RR, some associated with enhanced viral replication efficiency, other resulting in multiplication of binding sites for cellular transcription factors.

Objectives To evaluate the accuracy of the QuantiFERON-TB Gold assay (QFT-IT) in children with suspected active or latent TB infection (LTBI). Methods A retrospective study was conducted on 621 children (0–14 years old) evaluated for TB infection or disease. Following clinical assessment, children were tested with the QFT-IT assay. Results Among the 140 active TB suspects, we identified 19 cases of active disease. The overall sensitivity for active TB was 87.5%, ranging from 62.5% in children 25–36 months old to 100% in children older than 49 months. The overall specificity for active TB was 93.6%. Among the 481 children tested for LTBI screening, 38 scored positive and all but 2 had at least one risk factor for TB infection. Among the 26 children with indeterminate results, bacterial, viral or fungal pneumonia were later diagnosed in 11 (42.3%) cases and non-TB related extra-pulmonary infections in 12 (46.1%). Conclusions Our results indicate that the children's response to QFT-IT associates to active TB and risk factors for LTBI. Moreover, we show that mitogen response is also found in children of 1 year of age, providing support for QFT-IT use also in young children.

For decades, adjuvant hormonal therapy has become the standard treatment of patients with estrogen receptor-positive breast cancer. Currently, the drugs available are GnRH agonists, selective estrogen receptor modulators, and aromatase inhibitors. The use of GnRH agonists represents a potentially reversible treatment that can restore ovarian function after chemotherapy. In premenopausal women, systemic therapy based on selective estrogen receptor modulators administration (e.g., tamoxifen) usually represents the standard adjuvant treatment. There are not sufficient data to recommend the routine addition of GnRH agonists to other endocrine therapies. In postmenopausal women, the disease-free survival was significantly prolonged in patients treated with aromatase inhibitor compared with those treated with tamoxifen, but the survival benefit was modest. Better results were obtained when the two drugs were administered sequentially. According to the ASCO guidelines, after 5 years of tamoxifen treatment, either tamoxifen or aromatase inhibitors therapy should be suggested for an additional 5 years. Unfortunately, most adverse events are consistent with estrogen deprivation and are common to all therapies, and the cumulative toxicity causes discontinuation and nonadherence to therapy in up to 50% of patients. Switching tamoxifen to an aromatase inhibitor may reduce adverse event incidence. Molecular-targeted therapy is useful in patients with advanced, relapsed or hormonal therapy-resistant tumors, usually as second- or third-line treatment. These drugs are usually added to aromatase inhibitors; however, currently, they have not yet been used in patients with early breast cancer.

Background Osteopetrosis or Albers-Schonberg's disease is a heterogeneous group of rare hereditary troubles of the bone characterized by bone sclerosis due to an alteration of the bone reabsorption mediated by osteoclasts. The defect in the osteoclastic activity is responsible for complete or partial medullary cavities occlusion, with consequent reduced hemopoiesis, and for the excessive fragility of the affected bone segments. Case report We reported the case of a young man of 31 years affected by osteopetrosis in which a small cell lung cancer developed. Results Small cell lung cancer is a particularly rare neoplasm in the young, and even though it is highly sensitive to chemotherapeutic treatment its prognosis remains poor. The greatest clinical problem connected with chemotherapeutic treatment of patients affected by osteopetrosis is the variability of the reduction of their bone marrow reserve, which could expose them to an excessive hematological toxicity caused by the therapy. Conclusions The adoption of suitable prophylactic measures, such as the use of growth factors and drugs selected in relation to their toxicity or given in reduced doses, should be appropriately considered in these subjects.

In this study, we set up a real-time reverse transcriptase PCR assay to measure the relative amounts of brkA transcripts in 50 Bordetella isolates. The results suggested that brkA expression is strain dependent and its level may play a role in determining the serum resistance or susceptibility phenotype. Pertussis immunocompetent sera were unable to kill Bordetella parapertussis via complement deposition.

Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues.

We report that hbhA is differentially regulated during Mycobacterium tuberculosis infection. Upregulation was observed in epithelial cell infection but not in macrophage infection and in the lungs but not in the spleens of infected mice, and it was greater during the early steps of infection, when bacilli disseminate from the site of primary infection.

In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell envelope-related processes. Importantly, a sigL - rslA mutant strain was significantly attenuated in a mouse model of infection.

Introduction: HIV infection is considered a risk factor for severe outcomes of influenza A(H1N1)v infection. However, data on immune response against influenza A(H1N1)v virus in HIV-infected patients are lacking. Methodology: Data from seven HIV-positive and 14 HIV-negative patients infected with A(H1N1)v and from 23 HIV-positive and six HIV-negative asymptomatic controls were analyzed to evaluate the clinical picture, A(H1N1)v viral shedding, and the immune response against the virus. Results: Patients displayed mainly upper respiratory tract diseases (57.1%), while pneumonia was diagnosed only in HIV-negative patients (23.8% of subjects, of which 4.8% required intensive care unit admission). At day seven, 29% of HIV-infected patients were still positive for A(H1N1)v by RT-PCR on nasopharyngeal swabs. Interestingly, a persistence of CXCL10 secretion at high level and lower IL-6 levels was observed in HIV-positive subjects. The geometric mean haemagglutination inhibition titer (HI-GMT) and anti-influenza IgM levels were lower in HIV-positive individuals while anti-influenza IgG levels remained similar in the two groups. Conclusions: The immune impairment due to HIV infection could affect A(H1N1)v clearance and could lead to a lower antibody response and a persistent secretion of CXCL10 at high levels. However, the lower IL-6 secretion and treatment with highly active antiretroviral therapy (HAART) could result in a milder clinical picture.

Objectives We evaluated a new approach for the rapid detection of clarithromycin resistance in Helicobacter pylori, based on PCR and denaturing HPLC (DHPLC). Methods A 180 bp fragment of the 23S rRNA gene was amplified using DNA from 81 clinical H. pylori isolates (51 isolates were shown to be resistant to clarithromycin by Etest), and, directly, from 101 gastric biopsies from patients with digestive diseases, who were infected with H. pylori as assessed by a 13C-urea breath test, histology and/or culture. DHPLC was used to detect mutations in all the PCR products. Results DHPLC profiles for the 30 susceptible isolates all showed homoduplex peaks; the resistant isolates consistently generated heteroduplex peaks that were easily distinguishable from the wild-type H. pylori reference strain. Sequencing revealed point mutations in all the resistant isolates. Overall, five different mutations were detected. Four of these mutations (A2142G, A2142C, A2143G and T2182C) are known to be associated with clarithromycin resistance; the remaining mutation (C2195T) has not been previously described. This novel single-base substitution was found in combination with the common mutation A2143G. Of the biopsies tested, 25 specimens generated heteroduplexes due to sequence alterations (mutation A2142G, A2142C or A2143G). In one of these specimens, A2143G was found together with the novel mutation T2221C; in another, a mixture of wild-type and mutant (A2143G) sequences was detected. For 20 culture-positive out of the 25 biopsies DHPLC results confirmed the presence of clarithromycin resistance. Conclusions Our results suggest that the PCR-DHPLC assay is a valid tool for rapid assessment of clarithromycin resistance in H. pylori and that in the future it could be used directly on biopsy specimens, avoiding the need for culture-based methods.

The subtype of the Saccharomyces cerevisiae yeast species known as S. cerevisiae Hansen CBS 5926 was formerly believed to be a separate species, Saccharomyces boulardii. It is widely considered non-pathogenic and is used as a probiotic agent for treatment and prevention of diarrhea. The biological properties of Saccharomyces spp. show considerable intraspecies variability and the beneficial properties of probiotic yeasts are considered strain-specific. Septicemia and fungemia caused by S. boulardii have recently been described in both immunocompromised and immunocompetent patients receiving biotherapy with this yeast. It cannot be distinguished from other S. cerevisiae strains by phenotypic criteria, so identification of these infections requires molecular typing. To identify the most effective approach for distinguishing S. boulardii, we typed 35 isolates of S. cerevisiae, of which 27 were from various clinical specimens and 8 were isolates of S. boulardii (6 obtained from probiotic preparations and 2 from clinical specimens) using four different molecular methods, two based on PCR-restriction enzyme analysis or sequencing of rDNA spacer regions, the third based on microsatellite polymorphism analysis of the S. cerevisiae genes YKL139w and YLR177w, and the last based on hybridization analysis with retrotransposon Ty917. Several clinical isolates appeared to be identical to one or more other isolates with the first three methods used, whereas with the Ty917 hybridization method all of the isolates tested appeared to be very heterogeneous. The eight S. boulardii isolates were clearly distinguishable from the clinical S. cerevisiae isolates only with Ty917 hybridization and microsatellite DNA analyses. In the latter method, the eight S. boulardii isolates exhibited an allelic variant at one of loci tested that was not shared with any other strain. Our results suggest that microsatellite polymorphism analysis of the YKL139w and YLR177w genes, as well as the analysis by Ty917 hybridization, are the most useful tools for a correct identification of S. boulardii strains.

Sardinia is an island community of 1.6 million people. There are also about 3.5 million sheep and one hundred thousand cattle in which Johne's disease and Mycobacterium avium subspecies paratuberculosis infection are endemic. The present study was designed to determine what proportion of people in Sardinia attending for ileocolonoscopy with or without Crohn's disease were infected with this pathogen.Mycobacterium avium subspecies paratuberculosis was detected by IS900 PCR on DNA extracts of fresh intestinal mucosal biopsies as well as by isolation in culture using supplemented MGIT media followed by PCR with amplicon sequencing.Twenty five patients (83.3%) with Crohn's disease and 3 control patients (10.3%) were IS900 PCR positive (p = 0.000001; Odds ratio 43.3). Mycobacterium avium subspecies paratuberculosis grew in cultures from 19 Crohn's patients (63.3%) and from 3 control patients (10.3%) (p = 0.00001; Odds ratio 14.9). All patients positive by culture had previously been positive by PCR. Mycobacterium avium subspecies paratuberculosis first appeared in the liquid cultures in a Ziehl Neelsen (ZN) staining negative form and partially reverted through a rhodamine-auramine positive staining form to the classical ZN positive form. This resulted in a stable mixed culture of all 3 forms illustrating the phenotypic versatility of these complex chronic enteric pathogens.Mycobacterium avium subspecies paratuberculosis was detected in the majority of Sardinian Crohn's disease patients. The finding of the organism colonizing a proportion of people without Crohn's disease is consistent with what occurs in other conditions caused by a primary bacterial pathogen in susceptible hosts.

The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.

The Italian Epidemiological Survey evaluated antibiotic susceptibility of non-fermentative Gram-negative bacilli isolated from inpatient respiratory-tract specimens collected throughout Italy during 1997-1999. The minimal inhibitory concentrations of 14 antibiotics for 1474 Pseudomonas aeruginosa strains, 307 Stenotrophomonas maltophilia strains and 114 Acinetobacter baumannii strains were determined in 57 clinical microbiology laboratories by means of a standardised micro-dilution method. The most active drugs against P. aeruginosa isolates were meropenem (81% susceptible) and amikacin (80% susceptible). Imipenem and meropenem proved to be the only agents active against A. baumannii isolates, although 13 and 16%, respectively, of strains were resistant to these drugs. Trimethoprim-sulphamethoxazole (TMP-SMZ) showed activity only against S. maltophilia isolates (83% susceptible). A total of 185 multidrug-resistant P. aeruginosa isolates (resistant to piperacillin, ceftazidime, gentamicin, and imipenem) were found. Resistance rates and trends showed consistent regional variations, including sharp increases from 1997 to 1999 in imipenem resistance among P. aeruginosa isolates from central and southern Italy.

Background Cardiopulmonary bypass provokes a systemic inflammatory reaction that, in 1% to 2% of all cases, leads to multiorgan disfunction. The aim of this study was to evaluate the possible role of the intestine in the pathogenesis and development of this reaction. Methods Eleven selected patients scheduled for elective coronary artery bypass graft surgery were enrolled in a open, prospective clinical study. Gastric tonometry, chromium-labeled test and double sugar intestinal absorption tests, polymerase chain reaction microbial DNA test, and measurement of cytokines and transcriptional factor (nuclear factor κB) activation were performed. Results During the postoperative period, gastric pH remained stable (range,7.2 to 7.3). The partial pressure for carbon dioxide gradient between the gastric mucosa and arterial blood increased significantly (from 1 to 23 mm Hg), peaking in the sixth postoperative hour. Interleukin 6 increased significantly over basal levels, peaking 3 hours after cardiopulmonary bypass (96.3 versus 24 pg/mL). Nuclear factor κB never reached levels higher than those observed after lipopolysaccharide stimulation. Escherichia coli translocation was documented in 10 patients: in eight cases from removal of aortic cross-clamps and in two cases from the first postoperative hour. With respect to basal value (6.4%), the urine collection revealed a significant increase in excretion of the radioisotope during the first 24 hours after surgery (39.1%), although there were no significant variations with the double sugar test. Conclusions The results obtained showed a correlation between the damage of the gastrointestinal mucosa, subsequent increased permeability, E coli bacteremia, and the activation of a self-limited inflammatory response in the absence of significant macrocirculatory changes and postoperative complications.

Methyl mercury (MeHg) and metallic Hg are well known as neurotoxic agents. Dental amalgam contributes significantly to elemental Hg vapour exposure in the general population. There is little information about Hg concentration in human amniotic fluid (AF) of pregnant women and its potential toxic effect on the fetuses.Primary to assess the relationship between the presence of detectable mercury (Hg) concentration in human AF, number and surface areas of amalgam fillings of pregnant women; secondary to analyse their obstetric history and perinatal complications.Seventy-two pregnant women took part in this prospective study. One dentist recorded the dental status, presence, number and surface areas of amalgam fillings. Total Hg concentration in AF was determined in digested samples using automatic cold vapour atomic absorption equipment. The detection limit of Hg in AF, determined from blank readings, was 0.08 ng/ml. To estimate the dependence of the explanatory variables (such as number and surface areas of amalgam fillings, fish consumption, presence of liver or neurological diseases and smoking habits) on mercury concentration several linear regression models were built up. Stepwise logistic regression procedures were running on total sample and on patients with at least one amalgam filling (Positive Filling group = PF). Principal component analysis (PCA) provided two factors, which explained for more the 60% of the variance among the variables.The overall mean Hg concentration in AF among all patients was 0.37+/-0.49 ng/ml. Nineteen (26.4%) women had a Hg concentration0.08 ng/ml (Hg negative group). In 53 (73.6%) patients, with a concentrationor = 0.08 ng/ml (Hg positive group), the mean value of Hg was 0.49+/-0.52 ng/ml. The average number of amalgam fillings was 2.26 +/- 3.19 in the Hg negative group and 5.32+/-3.03 in the Hg positive group (ANOVA one-way p=0.04). A dependence of mercury concentration on number of amalgam fillings (p=0.03), surface area of the amalgam fillings (p=0.04) and fish consumption (p=0.04) was observed but not at a significant level. In stepwise logistic procedure the number of amalgam fillings gave a contribution to the model (p=0.04), although null value was included in the confidence intervals. We observed no statistically significant differences (chi2 test) among the patients with a Hg concentration0.08 ng/ml (n=19) and those with a concentrationor = 0.08 (n=53) with regard to obstetric history and perinatal complications.Number and surface areas of amalgam fillings influenced positively Hg concentration in AF but not at a significant level. Moreover Hg levels detected in AF were low and no adverse outcomes were observed through pregnancies and in the newborns.

Staphylococci are an increasing cause of bloodstream infections. Rapid reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment. We evaluated the ability of the VITEK 2 system (bioMérieux, Inc, Hazelwood, Mo.) to identify these organisms rapidly and accurately. A total of 405 clinically relevant nonduplicate staphylococcal isolates ( Staphylococcus aureus , n = 130; coagulase-negative staphylococci, n = 275) collected from blood cultures were tested. VITEK 2 results were considered correct when they were identical to those furnished by the comparison method based on the ID 32 STAPH system (bioMérieux, Marcy l'Etoile, France) plus supplementary manual testing. When discrepancies occurred, isolate identity was verified by molecular typing. The VITEK 2 correctly identified 387 (95.6%) isolates at the species level: 379 (including all but one [99.2%] of 130 S. aureus isolates and 249 of 275 [90.5%] coagulase-negative isolates) were identified by the automated reading; for the other eight, supplemental tests suggested by the manufacturer had to be used. Only one strain (0.2%) was misidentified ( Staphylococcus hominis as Staphylococcus epidermidis ), and four (1%), all S. epidermidis , were not identified. For the remaining 13 strains (including 10 S. hominis ), the VITEK 2 system was unable to discriminate among two species, and no supplemental tests were suggested for conclusive identification. Over 90% of results were obtained within 4 h. These results suggest that the VITEK 2 system can provide rapid, accurate, and reliable species-level identification of staphylococci responsible for bloodstream infections, although there is room for improvement in the identification of certain coagulase-negative species, especially S. hominis .

Summary Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ- cal meningitis, the most frequently encountered life- threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans , a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridiza- tion procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 ( CnAFR1 ). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced suscepti- bility of the knock-out mutant cnafr1 against flucona- zole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in flucona- zole resistance of C. neoformans . Our findings there- fore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.

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.

OBJECTIVES To define the incidence, risk factors and short-term predictors of mortality of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia in HIV-infected patients. PATIENTS AND METHODS All HIV-infected subjects with S. aureus bacteraemia were consecutively enrolled in a case-control study between January 1, 1991 and December 31, 2000 and prospectively followed up. RESULTS In the study period, 129 of 419 (31%) HIV-infected patients with bacteraemia had a diagnosis of S. aureus bacteraemia. The comparative analysis of incidence of S. aureus bacteraemia in the period 1991-96 and 1997-2000 showed a significant decrease (P < 0.001). The same trend was observed for MRSA bacteraemia (P = 0.002). The analysis of antimicrobial resistance showed that among 129 S. aureus strains, 88 (68%) were methicillin susceptible and 41 (32%) were methicillin resistant. The majority of MRSA bacteraemia was hospital acquired (78%). Previous administration of beta-lactams (P < 0.001), multiple previous hospital admissions (P < 0.001) and low numbers of CD4+ peripheral cells (P < 0.001) were found to be independent risk factors of methicillin resistance at multivariate analysis. The mortality rate was 34% in the cases and 11% in the controls (P = 0.002). Multivariate analysis indicated that a high Acute Physiology and Chronic Health Evaluation (APACHE) III score (P < 0.001) and high HIV viraemia (P = 0.02), but not methicillin resistance, predicted an increased risk of death in patients with S. aureus bacteraemia. CONCLUSION Individual exposure to beta-lactams, in association with a history of multiple hospitalizations and low CD4+ cell number, plays a pivotal role as a risk factor for the development of methicillin resistance in HIV-infected patients. Methicillin resistance does not influence the outcome of S. aureus bacteraemia when included in a multivariate analysis.

We describe an outbreak ofCandida albicanssystemic infection involving five premature infants in a neonatal intensive care unit. Molecular and epidemiologic characterization of allC. albicansisolates was performed by DNA fingerprinting with the 27A probe. This genotypic analysis demonstrated that the isolates were identical, providing evidence for the circulation of a uniqueC. albicansstrain.

In the present article we report cytomegalovirus (CMV) DNA localization in the inner ear of a 15-month-old deaf boy 1 month after a virologically documented primary infection. CMV DNA retrieval was possible thanks to polymerase chain reaction analysis of the perilymph collected at cochlear implant surgery. To the authors' knowledge this is the first demonstration of CMV persistence in the cochlea of an immunocompetent subject after an acquired infection. Laryngoscope, 2011