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Summary Objectives To identify risk factors for mortality in patients suffering from hematological malignancies with concurrent bacteremia caused by Escherichia coli. Particular attention was focused on defining the impact of extended-spectrum-β-lactamase (ESBL) production and fluoroquinolone resistance by the bacterial isolates on mortality. Materials and methods A retrospective eight-year cohort study design was employed. The outcome measured was death within 30 days of the first positive blood culture. Survivor and non-survivor subgroups were compared to identify predictors of mortality. Results A total of 62 episodes of bacteremia caused by E. coli were analyzed. The overall incidences of ESBL production and fluoroquinolone resistance were 41.9% and 62.9%, respectively. The overall 30-day mortality rate was 20.9% (13/62). In a multivariate analysis, significant predictors of mortality were inadequate initial antimicrobial therapy (OR = 14.96, 95% CI 1.95–114.51; P = 0.009), infection caused by ESBL-producing isolates (OR = 8.84, 95% CI 1.48–52.91; P = 0.01), and prolonged neutropenia (OR = 8.10, 95% CI 1.29–50.57; P = 0.02). Conclusions Sound knowledge of the local distribution of pathogens and their susceptibility patterns and prompt initiation of effective antimicrobial treatment are essential in patients suffering from hematological malignancies with BSIs caused by E. coli.

Accurate assessment of risk factors for nosocomial acquisition of colonization by antibiotic-resistant bacteria (ARB) is often confounded by scarce data on antibiotic use. A 12-month, nested, multicenter cohort study was conducted. Target ARB were methicillin (meticillin)-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and ciprofloxacin-resistant Pseudomonas aeruginosa (CR-PA). Nares and rectal swabs were obtained before and after starting antibiotics. Pulsed-field gel electrophoresis was done to define genetic relatedness of the strains. Primary outcomes were (i) the mean time, in days, for acquisition of target ARB colonization in patients previously not colonized; (ii) the rate of acquisition per 1,000 antibiotic-days according to different classes of antibiotics; (iii) the rate of infection caused by the same bacteria as those previously isolated in screening samples; and (iv) the risk factors for ARB acquisition. In total, 6,245 swabs from 864 inpatients were processed. The rate of acquisition was 3%, 2%, and 1% for MRSA, VRE, and CR-PA, respectively. The rate of acquisition of ARB per 1,000 antibiotic-days was 14 for carbapenems, 9 for glycopeptides, and 6 for broad-spectrum cephalosporins and quinolones. The highest rates of acquisition were observed for carbapenems in dialyzed and diabetic patients. Four risk factors were independently associated with acquisition of target ARB: use of carbapenems, age of >70 years, hospitalization for >16 days, and human immunodeficiency virus infection. During the 30-day follow-up, 4 among 42 patients newly colonized by ARB (9%) suffered from an infection due to the same bacteria as those isolated in a previous screening sample. Colonizing and infecting strains from single patients were genotypically identical. Identifying ARB colonization early during antibiotic therapy could target a high-risk hospitalized population that may benefit from intervention to decrease the risk of subsequent nosocomial infections.

Background and objectives: Bacterial-derived DNA fragments (BDNAs) have been shown to be present in dialysis fluid, to pass through dialyzer membranes, and to induce IL-6 (IL-6) in mononuclear cells. The present study aimed at assessing the eventual presence of BDNAs in the blood of hemodialysis (HD) patients and if this is associated with markers of chronic inflammation. Design, setting, participants, & measurements: Fifty-eight HD patients and 30 controls were included in the study. A blood sample was collected from a peripheral vein and from the central venous catheter (CVC) or the arteriovenous fistula (AVF) and examined for presence of BDNAs by 16S rRNA gene PCR amplification, bacterial growth, and measurement of C-reactive protein and IL-6. Thirty minutes after the start of HD, a sample of dialysis fluid was collected before the entry into and at the exit of the dialyzer and examined for presence of BDNAs. Results: Controls had negative blood cultures and absence of blood BDNAs. All HD patients had negative blood cultures, but in 12 (20.7%), BDNAs were present in the whole blood. In five of the latter, BDNAs were also found in the dialysis fluid. C-reactive protein serum levels (mg/L) were significantly higher in patients with than in those without BDNAs. Likewise, IL-6 serum levels (pg/ml) were significantly higher in patients with BDNA than in those without. Conclusions: Circulating BDNAs are associated with higher levels of C-reactive protein and IL-6 in HD patients. Clin J Am Soc Nephrol 4: 379 –385, 2009. doi: 10.2215/CJN.03490708

We conducted a retrospective cohort study to identify risk factors for mortality in a large cohort of hematologic patients with bacteremia. From 2000 through 2005, bacteremia was diagnosed in 217 patients with hematologic malignancies. The infections were caused only by Gram-positive organisms in 57.1% (124/217) cases and only by Gram-negative bacteria in 37.8% (82/217); the remaining 5.1% (11/217) were polymicrobial. The overall 30-day mortality rate was 20.3% (44/217). In multivariate analysis, significant predictors of mortality were prolonged neutropenia (P0.001), acute renal failure (P = 0.002), nosocomial bacteremia (P = 0.009), age55 years (P = 0.007), and monomicrobial bacteremia due to antibiotic-resistant Gram-negative bacteria (P = 0.009). Reducing fatal outcomes associated with bacteremia in patients with hematologic malignancies is a challenge, and the emergence of resistance to the antimicrobials widely used in this setting is of great concern. Future infection trends must be carefully monitored and treatment guidelines adjusted accordingly.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different “hot spots,” were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions., Author Summary Candida glabrata is a yeast causing several diseases in humans and especially in immuno-compromised people. C. glabrata infections are treated with antifungal agents, however the use of some agents, for example azoles, is associated with the development of resistance. In this yeast species, azole resistance is mediated almost exclusively by ATP Binding Cassette (ABC) multidrug transporters. Their overexpression results in enhanced efflux of azoles and thus generates resistance. Regulation of ABC transporters is therefore of pivotal importance to understanding azole resistance. In C. glabrata, the expression of ABC transporters is mediated by a zinc finger transcription factor called CgPDR1. Gain of function (GOF) mutations in CgPDR1 result in high ABC transporter expression. In this study, we investigated the occurrence of GOF mutations in a large collection of azole-resistant isolates and found a high variety of mutations localized in three distinct domains of CgPDR1. We found that these mutations are not only associated with resistance but also enhanced virulence and fitness of C. glabrata in animal models. Our study provides for the first time evidence that mutations causing antifungal resistance can also provide a selective advantage under host conditions and thus highlights the need of carefully monitoring resistance in this pathogen.

Candida surveillance cultures were obtained from 51 neonatal intensive care unit patients. Sixteen infants showing positive cultures developed subsequent Candida infection, whereas it did not occur for the 35 infants with negative cultures. Fifteen of 16 infants (

In this study, we characterized the humoral responses in cattle of Sardinia. The animals were divided into three groups: 1) 28 cattle infected with Mycobacterium bovis; 2) 48 cattle from herds in which foci of infection was notified; 3) 50 cattle from herds that were TB-free. Levels of IgG antibody were measured against the following antigens of M. tuberculosis: Heparin-Binding-Haemagglutin (HBHA), Ag85B, PPE44, and PE_PGRS33 to investigate their potential to diagnose TB in animals. Our results indicated that HBHA is a potential candidate for the development of a serological assay for rapid diagnosis of cattle infected with M. bovis.

Background: The multidrug-resistant (MDR) Acinetobacter baumannii calcoaceticus complex (Abc) has emerged as an important cause of nosocomial infections. The aims of the study were to evaluate risk factors for MDR-Abc in intensive care units (ICUs) as well as in medical and surgical wards, to define the likelihood ratios (LRs) of risk factors and to determine if risk factors differ depending on whether colonization or infections are considered. Methods: Two prospective matched case‐control studies were performed. MDR-Abc was defined as a strain resistant to four or more classes of antibiotics. The two case groups included patients with MDR-Abc infections or colonization. Controls were selected among patients not harbouring Abc. Matching criteria were the number of days from admission to MDR-Abc isolation among cases and the duration of hospitalization among controls. Results: Overall, 514 patients were included in the study. One hundred and thirty-seven patients were infected and 120 colonized. A Charlson score >3 and previous methicillin-resistant Staphylococcus aureus isolation and b-lactam use were independent risk factors for colonization and infection. Bedridden status and previous ICU admission were associated with colonization, while the presence of a central venous catheter and surgery were related to infection. The analysis of LRs showed an association between the presence of more than two risk factors and colonization or infection. The highest predicting value was observed for the presence of more than two risk factors and colonization in patients with no history of ICU admission. Conclusions: This study provides novel information that can be used to identify interventions for different stages of the spread of MDR-Abc.

Cyclospora cayetanensis is a coccidian agent of chronic diarrhea in humans with a worldwide distribution. We report the first documented case of acquired Cyclosporiasis in Italy. The patient was an immunocompetent woman with no recent history of travel outside the country. Microscopy detected Cyclospora oocysts in a feces sample. PCR detected the pathogen in a second sample, which had tested negative by microscopy. The patient was investigated to detect other microorganisms in feces, such as Salmonella spp., Shigella spp, Campylobacter spp., Yersinia spp, and enteroviruses: all were negative. All symptoms disappeared 72 h after the beginning of therapy.

Extended-spectrum-β-lactamase (ESBL)-producing strains of Escherichia coli are a significant cause of bloodstream infections (BSI) in hospitalized and nonhospitalized patients. We previously showed that delaying effective antimicrobial therapy in BSI caused by ESBL producers significantly increases mortality. The aim of this retrospective 7-year analysis was to identify risk factors for inadequate initial antimicrobial therapy (IIAT) (i.e., empirical treatment based on a drug to which the isolate had displayed in vitro resistance) for inpatients with BSI caused by ESBL-producing E. coli . Of the 129 patients considered, 56 (43.4%) received IIAT for 48 to 120 h (mean, 72 h). Independent risk factors for IIAT include an unknown BSI source (odds ratios [OR], 4.86; 95% confidence interval [CI], 1.98 to 11.91; P = 0.001), isolate coresistance to ≥3 antimicrobials (OR, 3.73; 95% CI, 1.58 to 8.83; P = 0.003), hospitalization during the 12 months preceding BSI onset (OR, 3.33; 95% CI, 1.42 to 7.79; P = 0.005), and antimicrobial therapy during the 3 months preceding BSI onset (OR, 2.65; 95% CI, 1.11 to 6.29; P = 0.02). IIAT was the strongest risk factor for 21-day mortality and significantly increased the length of hospitalization after BSI onset. Our results underscore the need for a systematic approach to the management of patients with serious infections by ESBL-producing E. coli . Such an approach should be based on sound, updated knowledge of local infectious-disease epidemiology, detailed analysis of the patient's history with emphasis on recent contact with the health care system, and aggressive attempts to identify the infectious focus that has given rise to the BSI.

Development of multigenic constructs expressing Mycobacterium tuberculosis (Mtb) antigens may be a strategy to obtain improved DNA vaccines against tuberculosis (TB). Several multigenic constructs expressing two or three Mtb antigens as fusion proteins were developed, both as tPA- and ubiquitin-fusion proteins. To demonstrate proper protein expression and intracellular turnover all multiantigens were tagged with the HA epitope and constructs were used to transfect rhabdomyosarcoma (RD) cells. Antigen expression was demonstrated by immunofluorescence using anti-HA antibodies. C57Bl/6 mice were immunized with selected constructs and protective activity was assessed following aerogenic challenge with Mtb. Several of these constructs induced a significant level of protection in the lung and in the spleen. Immunization with the construct expressing tPA85B-ES6 induced a level of protection that approached that provided by BCG. Immunization with a combination of these constructs induced levels of protection that were not superior to those elicited by a single combination, and immunization with a construct expressing five Mtb antigens could not provide an improved level of protection compared to tPA85B-ES6. We conclude that the activity of a DNA vaccine based on tPA85B-ES6 cannot be enhanced by broadening the antigen repertoire with other highly immunogenic secreted Mtb proteins.

Summary Our previous investigation on Candida glabrata azole-resistant isolates identified two isolates with unaltered expression of CgCDR1/CgCDR2, but with upregulation of another ATP-binding cassette trans- porter, CgSNQ2, which is a gene highly similar to ScSNQ2 from Saccharomyces cerevisiae. One of the two isolates (BPY55) was used here to elucidate this phenomenon. Disruption of CgSNQ2 in BPY55 decreased azole resistance, whereas reintroduction of the gene in a CgSNQ2 deletion mutant fully reversed this effect. Expression of CgSNQ2 in a S. cerevisiae strain lacking PDR5 mediated not only resistance to azoles but also to 4-nitroquinoline N-oxide, which is a ScSNQ2-specific substrate. A putative gain-of-function mutation, P822L, was iden- tified in CgPDR1 from BPY55. Disruption of CgPDR1 in BPY55 conferred enhanced azole susceptibility and eliminated CgSNQ2 expression, whereas intro- duction of the mutated allele in a susceptible strain where CgPDR1 had been disrupted conferred azole resistance and CgSNQ2 upregulation, indicating that CgSNQ2 was controlled by CgPDR1. Finally, CgSNQ2 was shown to be involved in the in vivo response to fluconazole. Together, our data first demonstrate that CgSNQ2 contributes to the devel- opment of CgPDR1-dependent azole resistance in C. glabrata. The overlapping in function and regula- tion between CgSNQ2 and ScSNQ2 further highlight the relationship between S. cerevisiae and C. glabrata.

International guidelines suggest that a high prevalence of meticillin-resistant Staphylococcus aureus (MRSA) infections should influence the use of vancomycin for surgical prophylaxis. In order to compare the efficacy and adverse effects of vancomycin versus cefazolin as antimicrobial prophylaxis for insertion of cerebrospinal fluid (CSF) shunts, a randomised prospective clinical trial was performed. Over a 16-month period, all consecutive adult patients who underwent CSF shunt insertion at a university hospital with a high prevalence of MRSA infections were included. Patients were randomly allocated to receive either vancomycin or cefazolin before surgery and followed-up for four weeks for the development of infections. Of the 176 patients included in the study, 88 received vancomycin and 88 cefazolin. Shunt infections were significantly less likely to be observed in patients who were on vancomycin prophylaxis (4% vs 14%; P=0.03). All isolated staphylococci were resistant to meticillin. Mortality of patients with post-surgical infections was higher in the cefazolin group (P=0.02). Our data suggest that use of vancomycin as prophylactic agent for cerebrospinal shunt placement reduces the rate of shunt infections in the context of high prevalence of MRSA.

Introduction Staphylococcus lugdunensis is an unusually virulent coagulase-negative staphylococcus that has rarely been implicated in central nervous system infections. Case presentation Two children hospitalized in the Neurosurgery Unit developed ventriculitis caused by methicillin-resistant Staphylococcus lugdunensis following placement of external ventriculostomy drains. The causative organisms were identified by molecular studies. The patients recovered without significant sequelae after high doses of intrathecal vancomycin. Conclusion Distinguishing Staphylococcus lugdunensis from other coagulase-negative staphylococcus species is crucial because it carries a substantial risk for severe central nervous system infections displayed by patients with implanted cerebrospinal fluid devices. Clinicians should not underestimate the importance of the isolation of this species from cerebrospinal fluid specimens.

Background In a recent multi-centre Italian survey (2003–2004), conducted in 45 laboratories throughout Italy with the aim of monitoring microorganisms responsible for severe infections and their antibiotic resistance, Acinetobacter baumannii was isolated from various wards of 9 hospitals as one of the most frequent pathogens. One hundred and seven clinically significant strains of A. baumannii isolates were included in this study to determine the in vitro activity of tigecycline and comparator agents. Methods Tests for the susceptibility to antibiotics were performed by the broth microdilution method as recommended by CLSI guidelines. The following antibiotics were tested: aztreonam, piperacillin/tazobactam, ampicillin/sulbactam, ceftazidime, cefepime, imipenem, meropenem tetracycline, doxycycline, tigecycline, gentamicin, amikacin, ciprofloxacin, colistin, and trimethoprim/sulphametoxazole. The PCR assay was used to determine the presence of OXA, VIM, or IMP genes in the carbapenem resistant strains. Results A. baumannii showed widespread resistance to ceftazidime, ciprofloxacin and aztreonam in more than 90% of the strains; resistance to imipenem and meropenem was 50 and 59% respectively, amikacin and gentamicin were both active against about 30% of the strains and colistin about 99%, with only one strain resistant. By comparison with tetracyclines, tigecycline and doxycycline showed a higher activity. In particular, tigecycline showed a MIC90 value of 2 mg/L and our strains displayed a unimodal distribution of susceptibility being indistinctly active against carbapenem-susceptible and resistant strains, these latter possessed OXA-type variant enzymes. Conclusion In conclusion, tigecycline had a good activity against the MDR A. baumannii strains while maintaining the same MIC90 of 2 mg/L against the carbapenem-resistant strains.

A certain number of isomeric 2-phenylquinoxalines N-oxides bearing substituents at position 6 7 were prepared in order to investigate anti-microbial activity. The effect of some electron—withdrawing groups seem to increase an anti-Trichomonas vaginalis activity in vitro. Structure elucidation of 6-acetyl-2-phenylquinoxaline was achieved by an unambiguous synthesis. 1H NMR spectra of N-oxides were examined in detail to establish structure of parent quinoxalines.

Immune-related disorders are frequently rampant in both developed and developing countries. It is speculated that such diseases probably reflect and connect to long-term effects of a change in lifestyle and thereby a reduced exposure to certain bacteria that have been inherently associated with human societies during most of mammalian evolution (3, 17). A very important group of bacteria among these organisms is saprophytic mycobacteria, which trigger regulatory immune cell populations (3, 17) such as cytokine-secreting and antigen-presenting cells. These immune cell populations are probably the deterrent to some autoimmune diseases such as type 1 (insulin-dependent) diabetes mellitus (T1DM). T1DM constitutes interactions of polygenic traits with not-well-known environmental factors, and it is not known what triggers autoimmunity to self-antigens such as those expressed in the pancreatic islets of Langerhans cells (5, 11). Drinking of cow's milk in childhood is assumed to be a risk factor for the development of this disease (17). The role of mycobacterial proteins that cross-react with epitopes of human cell surface molecules has been explored (3, 5). Human populations that lived hygienic lifestyles and therefore remained “sanitized” for decades might react aggressively to exposure to certain microbial communities such as Mycobacterium avium subsp. paratuberculosis. Due to the expansion of the dairy industry in developed countries as a result of modern animal breeding, the exposure of human populations to M. avium subsp. paratuberculosis has increased. Sardinia, Italy, is one such example, where intensive sheep farming is practiced and the sheep population constitutes more than four times the existing human population of this Mediterranean island. M. avium subsp. paratuberculosis bacilli have been notoriously known to trigger molecular mimicry (15, 23). It has long been a belief that genetic susceptibilities, epitope homologies, and endemic bacterial load in the environment might support the case for an infectious trigger, such as M. avium subsp. paratuberculosis, to be a causative agent of T1DM in genetically susceptible individuals (3, 8, 11, 17). However, their direct association with T1DM has remained largely elusive. Recent attempts (21) have been directed at the demonstration of clinically significant loads of M. avium subsp. paratuberculosis DNA in the blood of diabetes patients. However, it is essential to unravel the interaction of M. avium subsp. paratuberculosis bacilli with the host immune system to know if they are directly involved in the disease process. We attempted to test the association of M. avium subsp. paratuberculosis with T1DM in an endemic setting like Sardinia and demonstrate for the first time the presence of clinically significant humoral responses of T1DM patients to recombinant M. avium subsp. paratuberculosis antigens and whole-cell lysates.

Summary The PE family of Mycobacterium tuberculosis includes 98 proteins which share a highly homologous N-terminus sequence of about 110 amino acids (PE domain). Depending on the C-terminal domain, the PE family can be divided in three subfamilies, the largest of which is the PE_PGRS with 61 members. In this study, we determined the cellular localization of three PE proteins by cell fractionation and immunoelectron microscopy by expressing chimeric epitope-tagged recombinant proteins in Mycobacterium smegmatis. We demonstrate that the PE domain of PE_PGRS33 and PE11 (a protein constituted by the only PE domain) contains the information necessary for cell wall localization, and that they can be used as N-terminal fusion partners to deliver a sufficiently long C-terminus-linked protein domain on the mycobacterial cell surface. Indeed, we demonstrate that PE_PGRS33 and Rv3097c (a lipase belonging to the PE family) are surface exposed and localize in the mycobacterial cell wall. Moreover, we found that PE_PGRS33 is easily extractable by detergents suggesting its localization in the mycobacterial outer membrane. Beyond defining the cellular localization of these proteins, and a function for their PE domains, these data open the interesting possibility to construct recombinant mycobacteria expressing heterologous antigens on their surface for vaccine purposes.

Bloodstream infections (BSI) caused by extended-spectrum β-lactamase (ESBL)-producing organisms markedly increase the rates of treatment failure and death. We conducted a retrospective cohort analysis to identify risk factors for mortality in adult in-patients with BSI caused by ESBL-producing Enterobacteriaceae (ESBL-BSI). Particular attention was focused on defining the impact on the mortality of inadequate initial antimicrobial therapy (defined as the initiation of treatment with active antimicrobial agents >72 h after collection of the first positive blood culture). A total of 186 patients with ESBL-BSI caused by Escherichia coli ( n = 104), Klebsiella pneumoniae ( n = 58), or Proteus mirabilis ( n = 24) were identified by our microbiology laboratory from 1 January 1999 through 31 December 2004. The overall 21-day mortality rate was 38.2% (71 of 186). In multivariate analysis, significant predictors of mortality were inadequate initial antimicrobial therapy (odds ratio [OR] = 6.28; 95% confidence interval [CI] = 3.18 to 12.42; P < 0.001) and unidentified primary infection site (OR = 2.69; 95% CI = 1.38 to 5.27; P = 0.004). The inadequately treated patients (89 of 186 [47.8%]) had a threefold increase in mortality compared to the adequately treated group (59.5% versus 18.5%; OR = 2.38; 95% CI = 1.76 to 3.22; P < 0.001). The regimens most commonly classified as inadequate were based on oxyimino cephalosporin or fluoroquinolone therapy. Prompt initiation of effective antimicrobial treatment is essential in patients with ESBL-BSI, and empirical decisions must be based on a sound knowledge of the local distribution of pathogens and their susceptibility patterns.

A major barrier to successful viral suppression in HIV type 1 (HIV-1)-infected individuals is the emergence of virus resistant to antiretroviral drugs. We explored the evolution of genotypic drug resistance prevalence in treatment-failing patients from 1999 to 2005 in a clinical cohort.Prevalence of major International AIDS Society-USA HIV-1 drug resistance mutations was measured over calendar years in a population with treatment failure and undergoing resistance testing. Predictors of the presence of resistance mutations were analysed by logistic regression.Significant reductions of the prevalence of resistance to all three drug classes examined were observed. This was accompanied by a reduction in the proportion of treatment-failing patients. Independent predictors of drug resistance were the earlier calendar year, prior use of suboptimal nucleoside analogue therapy, male sex and higher CD4 levels at testing.In a single clinical cohort, we observed a decrease in the prevalence of resistance to all three examined antiretroviral drug classes over time. If this finding is confirmed in multicentre cohorts it may translate into reduced transmission of drug-resistant virus from treated patients.

A total of 750 clinical yeast isolates were evaluated by two identification systems, VITEK 2 and RapID Yeast Plus, using sequence analysis of the rRNA gene internal transcribed spacer regions as the reference method. The VITEK 2 and RapID systems correctly identified 737 (98.2%) and 716 (95.5%) isolates, respectively.

The most frequent agents of severe bacterial infections and their antibiotic susceptibility patterns were determined in patients admitted to 45 Italian hospitals over the years 2002-2003. The most common diagnoses were: sepsis (33.8%), pneumonia (9.4%), intravascular catheter-associated infections (9.3%) and ventilator-associated pneumonia (8.1%). Overall, 5115 bacterial isolates were identified from 4228 patients. Three bacterial species, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, accounted for more than 50% of the isolates. Other prevalent bacterial isolates were Staphylococcus epidermidis and Enterococcus faecalis, while Acinetobacter baumanii ranked third among all Intensive Care Unit (ICU) isolates. 7% of S. aureus had intermediate resistance to vancomycin. Although E. faecalis displayed no vancomycin resistance, 34% of vancomycin-resistant isolates were found among Enterococcus faecium, one of the highest rates found to date, emphasizing the difference between these two enterococcal species. All the Gram-positive pathogens were susceptible to linezolid, with the exception of approximately 2% of the enterococcal isolates that were intermediate with a minimum inhibitory concentration (MIC)=4 microg/ml. Almost 10% of Escherichia coli, 14% of Klebsiella pneumoniae, 22% of Serratia marcescens and 50% of Enterobacter cloacae were non-susceptible to cefotaxime. Amikacin was the most active antibiotic against P. aeruginosa that showed lack of susceptibility to ceftazidime, gentamicin, piperacillin and ciprofloxacin ranging from 20 to 35%. Finally, Acinetobacter baumanii showed a high level of resistance to all the antibiotics tested including imipenem (58%). The results obtained in this study, the first of its kind in Italy, offer indications for guiding empirical therapy and implementing specific interventions to fight antibiotic-resistant bacterial infections and their transmission in the hospital setting in Italy.

Leuconostoc species are emerging pathogens that can cause severe infections, particularly in immunocompromised patients. Using molecular methods, we identified Leuconostoc mesenteroides as the cause of a brain abscess which was successfully treated by surgery and antimicrobial treatment. This is the first report of brain abscess caused by this species.

Primary central nervous system lymphoma (PCNSL) is a major complication of the late stages of human immunodeficiency virus (HIV) disease. Epstein Barr virus (EBV) infection is the only genetic lesion consistently associated with this neoplasia. Recently, it has been proposed that the pathogenesis of AIDS-related PCNSL (AIDS-PCNSL) may be associated with infection by human herpesvirus-8/Kaposi's sarcoma associated herpesvirus (HHV-8/KSHV), although at present such association remains controversial. In order to conclusively assess the link between HHV-8/KSHV infection and AIDS-PCNSL, we performed a comprehensive study based on multiple molecular assays on cerebral tissues and cerebrospinal fluid (CSF) as well as specific immunologic assays on patients' sera. A well characterized panel of 33 Italian patients with AIDS-PCNSL and 13 controls with other HIV-related brain focal diseases from the same geographical area was analyzed. No signs of HHV-8/KSHV infection were detected in cerebral tissues by single-step PCR. Cerebral tissues of all AIDS-PCNSL scored negative for HHV-8/KSHV DNA sequences also by nested PCR, with the exception of one single patient who was simultaneously affected by Kaposi's sarcoma. All CSF samples analyzed were consistently devoid of HHV-8/KSHV sequences by molecular assays. By serologic assays, detecting both latent and lytic HHV-8/KSHV antigens, a specific immunoreactivity was observed in 16/33 (48%) AIDS-PCNSL and in 6/13 (46%) controls (P = 0.88). A significant correlation with HHV-8/KSHV serum reactivity was seen with a homosexual route of HIV transmission (P = 0.018), but not with the presence of AIDS-PCNSL. The results of our analysis conclusively assess that HHV-8/KSHV infection is not a feature of AIDS-PCNSL.

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene AFR1 in Cryptococcus neoformans is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of AFR1 in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of C. neoformans , strain BPY22; an afr1 mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an AFR1- overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the AFR1 gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of C. neoformans .

Bloodstream infections caused by extended-spectrum-β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates are a major concern for clinicians, since they markedly increase the rates of treatment failure and death. One hundred forty-seven patients with K. pneumoniae bloodstream infections were identified over a 5-year period (January 1999 to December 2003). The production of ESBLs in bloodstream isolates was evaluated by molecular methods. A retrospective case-case-control study was conducted to identify risk factors for the isolation of ESBL - producing K. pneumoniae or non-ESBL - producing K. pneumoniae isolates in blood cultures. Forty-eight cases infected with ESBL - producing K. pneumoniae isolates and 99 cases infected with non-ESBL - producing K. pneumoniae isolates were compared to controls. Risk factors for isolation of ESBL - producing K. pneumoniae isolates were exposure to antibiotic therapy (odds ratio [OR], 11.81; 95% confidence interval [CI], 2.72 to 51.08), age (OR, 1.14; 95% CI, 1.08 to 1.21), and length of hospitalization (OR, 1.10; 95% CI, 1.04 to 1.16). Independent determinants for isolation of non-ESBL - producing K. pneumoniae were previous urinary tract infection (OR, 8.50; 95% CI, 3.69 to 19.54) and length of hospitalization (OR, 1.07; 95% CI, 1.04 to 1.10). When the initial response was assessed at 72 h after antimicrobial therapy, the treatment failure rate for the ESBL - producing K. pneumoniae -infected group was almost twice as high as that of the non-ESBL - producing K. pneumoniae -infected group (31% versus 17%; OR, 2.19; 95% CI, 0.98 to 4.89). The 21-day mortality rate for all patients was 37% (54 of 147); it was 52% (25 of 48) for patients with ESBL - producing K. pneumoniae bloodstream infections and 29% (29 of 99) for patients with non-ESBL - producing K. pneumoniae bloodstream infections (OR, 2.62; 95% CI, 1.28 to 5.35). In summary, this investigation identifies epidemiological characteristics that distinguish ESBL - producing K. pneumoniae infections from non-ESBL - producing K. pneumoniae ESBL bloodstream infections.

Background: Bacterial biofilms identified in various medical devices used in otorhinolaryngology, including tympanostomy tubes, voice prostheses, and cochlear implants, can directly colonise mucosal tissues. The upper airways seem to be at high risk for this type of colonisation. Chronic and/or recurrent upper airway infections may be related to the complex structural and biochemical (quorum sensing) organisation of the biofilm which interferes with the activity of antibiotics (including those with proven in vitro efficacy), thus promoting the establishment of a chronic infection eradicable only by surgical treatment. Biofilm formation plays a role in upper respiratory infections: it not only explains the resistance of these infections to antibiotic therapy but it also represents an important element that contributes to the maintenance of a chronic inflammatory reaction.Objectives: To document the presence of biofilms in surgical tissue specimens from patients with recurrent infection diseases, and identify their possible role in the chronicity of these infectious processes.Method: We examined 32 surgical specimens from the upper respiratory tract (tonsils, adenoids, mucosa from the ethmoid and maxillary sinuses) of 28 patients (20 adults, eight children) with upper airway infections that had persisted despite repeated treatment with anti-inflammatory agents and antibiotics with demonstrated in vitro efficacy. Tissues were cultured using conventional methods and subjected to scanning electron microscopy for detection of biofilm formation.Results: Over 80 per cent (26/32; 81.3 per cent) of the tissue specimens were culture-positive. Bacterial biofilms (associated in most cases with coccoid bacteria) were observed in 65.6 per cent of the tissue samples.

Extended-spectrum beta-lactamases (ESBLs) are a large, rapidly evolving group of enzymes that confer resistance to oxyimino cephalosporins and monobactams and are inhibited by clavulanate. Rapid reliable detection of ESBL production is a prerequisite for successful infection management and for monitoring resistance trends and implementation of intervention strategies. We evaluated the performance of the new VITEK 2 ESBL test system (bioMérieux, Inc, Hazelwood, Mo.) in the identification of ESBL-producing Enterobacteriaceae isolates. We examined a total of 1,129 clinically relevant Enterobacteriaceae isolates (including 218 that had been previously characterized). The ESBL classification furnished by the VITEK 2 ESBL test system was concordant with that of the comparison method (molecular identification of beta-lactamase genes) for 1,121 (99.3%) of the 1,129 isolates evaluated. ESBL production was correctly detected in 306 of the 312 ESBL-producing organisms (sensitivity, 98.1%; positive predictive value, 99.3%). False-positive results emerged for 2 of the 817 ESBL-negative isolates (specificity, 99.7%; negative predictive value, 99.3%). VITEK 2 ESBL testing took 6 to 13 h (median, 7.5 h; mean ± SD, 8.2 ± 2.39 h). This automated short-incubation system appears to be a rapid and reliable tool for routine identification of ESBL-producing isolates of Enterobacteriaceae .

Background Streptococcus intermedius belongs to the Streptococcus anginosus group. It is part of the normal flora of the human mouth, but it can be etiologically associated with deep-site infections. Case presentation We present a case of deep neck infection complicating Streptococcus intermedius lymphadenitis, which developed in an immunocompetent 14-year-old boy with a history of recent dental work. The infection was ultimately eradicated by a combined medical and surgical approach. Conclusion Our report suggests that combined medical and surgical therapy is essential for the complete resolution of deep infections caused by Streptococcus intermedius. Molecular biological techniques can be useful in guiding the diagnostic investigation and providing insight into the possibility of occult abscesses, which are particularly common with Streptococcus intermedius infections.

PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to H2O2 challenge (PperR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.

The present study was performed to determine what proportion of people in Sardinia with or without Crohn's disease were infected with Mycobacterium avium subspecies paratuberculosis and had a preponderance of allelic variants of Nod2, an intracellular protein involved in Crohn's disease susceptibility. Genetic analysis of the alleles of the NOD2/CARD15 gene ( insC3020 , G908R, and R702W alleles), linked to susceptibility or genetic predisposition to Crohn's disease in humans, was carried out on specimens from 37 Crohn's disease patients and 34 patients without Crohn's disease. Our results show that more than 70 percent of people in Sardinia with Crohn's disease carry at least one of the susceptibility-associated NOD2/CARD15 alleles and were infected with Mycobacterium avium subspecies paratuberculosis .

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's Disease, a chronic granulomatous enteritis of ruminants. Recently, an association of MAP bacilli with Crohn's disease in humans has been proposed. Due to genetic similarities and serological cross-reactivity of the M. avium complex with other mycobacteria, functional analysis of species-specific proteins may allow new insights into the pathogenesis of mycobacterial diseases. We report production and molecular characterization of the recombinant HBHA from the MAP complex bacilli. The HBHA was expressed in Escherichia coli and Mycobacterium smegmatis using efficient expression vector systems. The recombinant HBHA was found to be immunogenic and therefore induced antibody responses in cattle against the MAP bacilli with a possible cross reactivity with M. bovis infection. The MAP complex HBHA was thus found to be a target of the host humoral responses in Johne's disease. The recombinant HBHA protein was also found to be adherent to the Caco2 cell lines in-vitro, a significant observation to understand possible virulence mechanisms. Since M. tuberculosis HBHA was earlier shown to be involved in dissemination of the tubercle bacilli, the immunogenicity and cytoadherent nature of this MAP protein possibly suggests functional promiscuity.

An iCycler iQ real-time PCR assay targeting 18S rRNA Aspergillus -specific sequences was developed for the diagnosis of invasive pulmonary aspergillosis (IPA). Positive findings were obtained for 18 of 20 (90%) bronchoalveolar lavage (BAL) fluid specimens from patients with probable or confirmed IPA and were obtained for none of the 24 BAL samples from patients with no clinical evidence of aspergillosis. These results were concordant with those of a nested PCR assay, which detected 90% of the patients with IPA, while galactomannan ELISA revealed positivity for 100% of these patients, suggesting that combined use of methods might improve the diagnosis of IPA.

The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene ( CgERG11 ), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata , of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2 , alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2 . Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11 , were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2 , were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1 -, CgCDR2 -, and CgSNQ2 -encoded efflux pumps might explain the azole resistance in our set of isolates.

Reactivities of human sera against selected recombinant Mycobacterium tuberculosis antigens were assessed by enzyme-linked immunosorbent assay. The results obtained indicate that patients with tuberculosis (TB) do not develop a strong humoral response against PE_PGRS and PPE proteins or against the Ag85B and heparin-binding hemagglutinin (HBHA) recombinant antigens. Conversely, purified methylated HBHA was strongly recognized by sera obtained from TB patients compared to controls.

In this study, a total of 204 Mycobacterium tuberculosis DNAs from Sicily ( n = 144) and Sardinia ( n = 60) were studied by three genotyping methods. Results were analyzed both within and across islands, to define the phylogeographical specificities of the genotypes, look for their diversity and infer a molecular evolutionary scenario. A strong link between geography and tuberculosis genotypes was observed in Sardinia. The results were also matched against a world-wide genetic diversity database to compare the population structure of the tubercle bacilli in the islands. Eight common genotypes between Sicily, Sardinia and continental Italy were found which underlines the influences of the Italian mainland on the population structure on the islands and vice versa. A unified evolutionary scenario of M. tuberculosis evolution was built using numerical taxonomy and maximum parsimony (MP) methods. The finding of multiple families of M. tuberculosis strains (S, T, LAM, Haarlem), their presumed links with the major genetic groups (MGG) of M. tuberculosis complex, supports the view of independent introduction of several ancestral genotypes in Sicily and in Sardinia. We conclude that the two PCR-based genotyping combination (spoligotyping–VNTR) is an excellent tool to reconstruct M. tuberculosis phylogeny, that may be used to construct global and local evolutionary scenarios of the M. tuberculosis complex. The results obtained are paradigmatic of the complex interplay that exists between epidemic dynamics and evolutionary genetics of M. tuberculosis.

We developed a new method based on the Nanochip microelectronic array technology for identification of various clinically relevant mycobacterial species. PCR-amplified rRNA genes obtained from 270 positive Mycobacteria Growth Indicator Tube cultures were successfully tested by hybridizing them with species-selective probes, and the results agreed with those of conventional identification methods. The system is rapid and accurate and opens new perspectives in clinical diagnostics.

The in-vitro activity of a 3-azinomethyl-rifamycin (SPA-S-565) against Chlamydia trachomatis was compared to that of rifampicin and of erythromycin. The compound showed excellent activity for standard strains as well as isolates from patients with sexually transmitted diseases, being more active than the other drugs tested. SPA-S-565 also showed a low frequency of emergence of resistance. Passage of standard strains at sub-inhibitory concentrations caused an increase in MIC values of rifampicin while those for SPA-S-565 remained unchanged.

Although alemtuzumab (campath-1H) has been successfully used in patients with untreated or previously treated chronic lymphocytic leukemia (CLL), a variable incidence of cytomegalovirus (CMV) reactivation has been described. No prospective reports currently provide results of the use of oral ganciclovir as pre-emptive therapy in patients with CMV reactivation during alemtuzumab treatment.We designed a prospective study in 12 patients with pretreated CLL with the aim of evaluating the incidence of CMV reactivation during alemtuzumab treatment and the role of oral ganciclovir as pre-emptive therapy and in preventing CMV organ disease.In the 12 CLL patients being treated with alemtuzumab, 8 patients (66%) had CMV reactivation, as detected by antigenemia and/or CMV DNA. No patient showed clinical evidence of CMV disease. The alemtuzumab was discontinued and the patients were immediately treated with oral ganciclovir 1000 mg tid. After a median of 14 days of antiviral therapy all patients achieved negative CMV polymerase chain reaction (PCR) assays and/or antigenemia. No patients showed further CMV reactivation up to the end of the study.CMV reactivaction, studied with periodic analysis of antigenemia and PCR, is frequent in previously treated CLL patients receiving alemtuzumab therapy although only sporadic cases of CMV disease have been reported. Using oral ganciclovir, the response to therapy was prompt, there was no progression to CMV disease, and no relevant clinical toxicity, thus sparing unnecessary hospitalization. Oral ganciclovir may be used as pre-emptive therapy in all patients who develop CMV reactivation during alemtuzumab treatment.

Objectives.To perform a 3-year, prospective surveillance program for legionnaires disease (LD) in a large university hospital in Rome, and to assess the usefulness of the hospital water monitoring program in predicting the risk of nosocomial LD.Methods.Samples from patients with new cases of nosocomial pneumonia were sent for legionella laboratory investigations. Meanwhile, water samples for bacteriological analysis were collected every 6 months from high- and medium-risk hospital wards (10 in total).Legionella pneumophilaisolates collected were serotyped and analyzed by pulsed-field gel electrophoresis.Results.From June 2001 through May 2004, the pneumonia surveillance identified one case of nosocomial LD among 43 cases of nosocomial pneumonia (2.3%). Environmental investigations detectedL. pneumophilain 12 (18.7%) of the 64 water samples, of which 50% belonged to serogroup 1. TheL. pneumophilacount and the percentage of positive locations never exceeded 102colony-forming units/L and 20%, respectively, except when the LD nosocomial case occurred (positive water samples, 40%; I. pneumophila count, 2colony-forming units/L). Genotyping showed 3 prevalent clones ofL. pneumophilain the water distribution network, of which one persisted over the 3 years. One clone contained 3 differentL. pneumophilaserogroups (2, 4, and 6).Conclusions.The low incidence of nosocomial cases of LD appears to be associated with a low percentage (2colony-forming units/L). An infection control system for nosocomial LD should, therefore, be based on both environmental and clinical surveillance, together with the appropriate maintenance of the hospital water distribution system.

Objective:To investigate an outbreak ofCandida parapsilosisbloodstream infections (BSIs) involving three patients admitted to a pediatric oncology unit between April and June 2002.Methods:After the third case was documented, cultures were performed of the hands of all medical and paramedical staff members in the pediatric oncology unit and of environmental surfaces in the rooms occupied by the three patients. Electrophoretic karyotyping with pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction were used to assess the genetic relatedness amongC. parapsilosisisolates.Results:The three cases ofC. parapsilosisBSI were diagnosed based on blood cultures performed during a 38-day period. Evidence of priorC. parapsilosiscolonization of the gastrointestinal tract was present in only the first case. Each patient had an indwelling central venous catheter (CVC), which was promptly removed, and semiquantitative catheter tip cultures also revealedC. parapsilosis. None of the 30 environmental cultures performed was positive forC. parapsilosis, but the fungus was isolated from the hands of 6 of the 20 nurses tested. Both molecular typing methods revealed identical DNA fingerprinting patterns for all 13 patient isolates (7 from blood, 3 from CVC tips, and 3 from the gastrointestinal tract) and for 5 of the 6 recovered from the nurses' hands.Conclusions:These findings suggest the possibility of cross-infection with a singleC. parapsilosisstrain that was transmitted (probably during CVC dressing changes) by nurses whose hands were colonized with it. The role of previous gastrointestinal colonization in the first case cannot be excluded.

In order to identify regulators of the oxidative stress response in Enterococcus faecalis , an important human pathogen, several genes annotated as coding for transcriptional regulators were inactivated by insertional mutagenesis. One mutant, affected in the ef2958 locus (designated hypR [hydrogen peroxide regulator]), appeared to be highly sensitive to oxidative challenge caused by hydrogen peroxide. Moreover, testing of the hypR mutant by using an in vivo-in vitro macrophage infection model resulted in a highly significant reduction in survival compared to the survival of parent strain JH2-2. Northern blot analyses were carried out with probes specific for genes encoding known antioxidant enzymes, and they showed that the ahpCF (alkyl hydro p eroxide reductase) transcript was expressed less in mutant cells. Mobility shift protein-DNA binding assays revealed that HypR regulated directly the expression of hypR itself and the ahpCF operon. Our combined results showed that HypR appeared to be directly involved in the expression of ahpCF genes under oxidative stress conditions and suggested that this regulator could contribute to the virulence of E. faecalis .

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.

We report the case of a 52-year-old male heart transplant recipient with a persistent localized subcutaneous infection by Scedosporium apiospermum. This form differs from the most common mycetoma by the absence of granules. The patient showed multiple nodules on the right hand that were surgically removed. Concomitantly, he received oral itraconazole, but the infection persisted for two years, and several surgical interventions were necessary to eradicate the infection. Our case demonstrates that a medical approach alone may be not sufficient to cure this fungal infection.

To investigate the epidemiological and clinical findings of extended-spectrum beta-lactamase (ESBL)-producing Providencia stuartii infections in a large Italian university hospital.All consecutive episodes of P. stuartii infection that occurred during 1999-2002 were included in the study. For each patient, we recorded the area of hospitalization and drug susceptibility of the P. stuartii strains. Patients with ESBL-producing P. stuartii infection were considered cases and those with non-ESBL-producing P. stuartii infection were used as controls.One hundred and sixteen (52%) out of 223 P. stuartii strains collected during the study period were found to be ESBL-producing. On the basis of PCR and DNA sequencing experiments, TEM-52 was identified in 87% of isolates and TEM-72 in 13%. All ESBL-producing P. stuartii infections were nosocomially acquired. The prevalence increased from 31% of P. stuartii infections in 1999 to 62% in 2002 (P = 0.04). All 116 strains were classified as ESBL-producing multidrug-resistant P. stuartii, since 88% of the isolates were cross-resistant to ciprofloxacin and amikacin and the other 12% were cross-resistant to ciprofloxacin and gentamicin. At logistic regression analysis, advanced age (P0.001), previous hospitalization (P0.01), neoplastic disease (P0.001) and previous antibiotic therapy (P0.001) were independent risk factors for the development of ESBL-producing infections.This 4 year surveillance of Providencia complaints clearly indicates that infections caused by ESBL-producing multidrug-resistant P. stuartii are an emerging problem.

Current treatment for Helicobacter pylori infections generally includes two or more antimicrobials (amoxicillin, clarithromycin, nitroimidazoles, tetracycline, etc.), but treatment fails in 10–20% of all cases, often because of drug resistance. Levofloxacin has been proposed as an alternative for these refractory infections. We examined 67 H. pylori isolates from patients unsuccessfully treated with amoxicillin, clarithromycin, metronidazole and levofloxacin. Minimum inhibitory concentrations determined with the epsilometer test revealed clarithromycin and metronidazole resistance in 91 and 82.1% of the isolates, respectively; 52 (77.6%) were resistant to both drugs. All 67 isolates were susceptible to amoxicillin and tetracycline. Fifty-two isolates had levofloxacin MICs of 0.01–2 mg/l; the remaining 15 (22.4%), all clarithromycin- and metronidazole-resistant, had MICs ≥ 8 mg/l. Levofloxacin may be an option for refractory H. pylori infections, but the choice should be based on in vitro susceptibility data, and physicians should consider local resistance patterns when treating these infections empirically.

A human recombinant monoclonal Fab fragment that specifically recognizes all the influenza A virus strains tested was produced in transformed Escherichia coli using the phage display technique. No strain of influenza B virus reacted with it. It was purified after four cycles of panning and by a single passage through an immunoaffinity column. About 1 mg of pure monoclonal antibody was obtained from 1 liter of culture medium in 3 working days. The Fab fragment reacted with a viral 27-kDa protein, which could reasonably be a matrix protein. Indirect immunofluorescence tests performed on virus-infected MDCK cells showed that this Fab fragment was at least equally efficient as other commercial monoclonal antibody-based systems in detecting influenza A viral infections. The potential advantages of human recombinant Fabs on murine monoclonal antibodies are discussed.