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Conventional diagnostic methods for bacterial meningitis are frequently not rapid or sensitive enough to guide initial antimicrobial therapy. Streptococcus pneumoniae is the most frequent and severe cause of community-acquired bacterial meningitis and treatment is complicated by the increasing prevalence of antimicrobial resistance to third-generation cephalosporins. We used a new rapid antigen test in the cerebrospinal fluid and urine of patients with suspected bacterial meningitis, and found it to be highly sensitive and specific for the detection of pneumococci. This test might help guide initial therapy for bacterial meningitis according to the local rates of pneumococcal antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Most attention is given to seasonal influenza and respiratory syncytial virus outbreaks, but the cumulative burden caused by other respiratory viruses (RV) is not widely considered. The aim of the present study is to describe the circulation of RV in the general population during six consecutive seasons from 2006 to 2012 in Catalonia, Spain. Cell culture, immunofluorescence and PCR-based assays were used for the RV laboratory-confirmation and influenza subtyping. Phylogenetic and molecular characterizations of viral haemagglutinin, partial neuraminidase and matrix 2 proteins were performed from a representative sampling of influenza viruses. A total of 6315 nasopharyngeal samples were collected, of which 64% were laboratory-confirmed, mainly as influenza A viruses and rhinoviruses. Results show the significant burden of viral aetiological agents in acute respiratory infection, particularly in the youngest cases. The study of influenza strains reveals their continuous evolution through either progressive mutations or by segment reassortments. Moreover, the predominant influenza B lineage was different from that included in the recommended vaccine in half of the studied seasons, supporting the formulation and use of a quadrivalent influenza vaccine. Regarding neuraminidase inhibitors resistance, with the exception of the 2007/08 H275Y seasonal A(H1N1) strains, no other circulating influenza strains carrying known resistance genetic markers were found. Moreover, all circulating A(H1N1)pdm09 and A(H3N2) strains finally became genetically resistant to adamantanes. A wide knowledge of the seasonality patterns of the RV in the general population is well-appreciated, but it is a challenge due to the unpredictable circulation of RV, highlighting the value of local and global RV surveillance., (Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objectives: To investigate the potential relationship between quinolone resistance and biofilm production in a collection of Salmonella enterica clinical isolates and in S. enterica serovar Typhimurium serial mutants with increasing resistance to ciprofloxacin., Methods: Nalidixic acid susceptibility and biofilm formation were assessed in a collection of 122 S. enterica clinical isolates. An in vitro quinolone-resistant mutant, 59-64, was obtained from a biofilm-producing and quinolone-susceptible clinical isolate, 59-wt, in a multistep selection process after increasing ciprofloxacin concentrations. The quinolone resistance mechanisms [target gene and multidrug resistance (MDR) regulatory mutations, MICs of several antibiotics, cell envelope protein analysis, real-time PCR and ciprofloxacin accumulation] were characterized for mutant strains. In addition, analysis of fitness, biofilm formation, rdar morphotype and expression of biofilm-related genes by real-time PCR were also determined., Results: Nalidixic acid-susceptible S. enterica strains were more prevalent in producing biofilm than the resistant counterparts. Strain 59-64 acquired five target gene mutations and showed an MDR phenotype. AcrAB and acrF overexpression were ruled out, whereas TolC did show increased expression in 59-64, which, in addition, accumulated less ciprofloxacin. Consistently, increased ramA expression was seen in 59-64 and attributed to a mutation within its promoter. Reduced biofilm production related to diminished csgB expression as well as reduced fitness was seen for 59-64, which was unable to form the rdar morphotype., Conclusions: Quinolone resistance acquisition may be associated with decreased production of biofilm due to lower csgB expression. Efflux, biofilm production and fitness seem to be interrelated., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Although particular attention is paid to influenza A and B virus isolates during influenza surveillance, influenza C virus (FLUCV) coexisted during the first influenza A (H1N1) 2009 pandemic wave during the 2009-2010 season. From 27 April 2009 to 9 May 2010, 12 strains of FLUCV were detected in specimens collected from 1713 nonhospitalized patients with upper respiratory tract illness using a molecular method. Half of the patients with FLUCV infection were older than 14 years. The most frequent symptoms were cough and fever, similar to other viral respiratory infections. Phylogenetic analysis of the hemagglutinin-esterase gene revealed that the strains belonged to the C/Kanagawa/1/76-related and C/Sao Paulo/378/82-related lineages, demonstrating their co-circulation in Catalonia. In addition to regular virological surveillance that provides information about the incidence and the exact role of FLUCV in acute viral respiratory infections in the general population, the genetic lineage identification offers additional data for epidemiological purposes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Prolonged viral excretion in immunocompromised hosts leads to long oseltamivir treatment and to the subsequent development of oseltamivir-resistant pandemic influenza virus selection. We report the selection and nasopharyngeal shedding kinetics of an oseltamivir-resistant strain in a hospitalized immunocompromised patient with prolonged influenza illness. Viral load quantification and genotyping methods were performed from 7 serial nasopharyngeal samples. Before initial oseltamivir treatment, the viral load was 5.78 log(10) copies/mL of sample and only wild-type virus population was detected. The nasopharyngeal viral load remained above the detection limit although there was a second course of oseltamivir treatment. Twelve days after the onset of symptoms, an oseltamivir-resistant strain was selected. After 12 days of inhaled zanamivir treatment, the patient was discharged asymptomatic. The study emphasizes the importance of viral load quantification and surveillance of emergence of resistant strains prospectively because the information provided has important implications in the clinical management of the patient., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The objective of this study was to determine whether the prevalence of Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations has changed since the introduction of combined antiretroviral therapy (cART) and whether the mutations are associated with poor outcome in Spanish HIV-1-infected patients with Pneumocystis pneumonia (PcP). We studied 167 PcP episodes in HIV-1-infected patients diagnosed during the pre-cART (1989-1995) and cART (2001-2004) periods. Molecular genotyping of DHPS was successfully performed in 98 patients (43 pre-cART and 55 cART). Seventeen patients (17/98, 17%; 95% confidence interval [CI], 10-25%) had mutations in the DHPS gene: 14 patients (14/43, 33%; 95% CI, 19-49%) from the pre-cART period and 3 patients (3/55, 5.5%; 95% CI, 1.3-16%) from the cART period (P < 0.01). In the multivariate analysis, the pre-cART period, previous PcP prophylaxis with sulfa drugs, and homosexuality as an HIV risk factor were found to be associated with a higher risk of presenting DHPS mutations. Overall, 95% of patients were treated with trimethoprim and sulfamethoxazole (TMP-SMX). In-hospital mortality was similar in patients with (out) mutations (6% versus 11%, P = 0.84). DHPS gene mutations were more common during the pre-cART period and were associated with previous sulfa exposure and homosexuality. However, their presence did not worsen prognosis of PcP. The response to TMP-SMX with therapeutic doses was successful in most cases.

This study evaluated the daptomycin activity against two methicillin-resistant Staphylococcus epidermidis (MRSE) clinical isolates with different vancomycin susceptibilities: MRSE-375, with a vancomycin MIC of 2 microg/ml, and NRS6, a glycopeptide-intermediate S. epidermidis (GISE) strain with a vancomycin MIC of 8 microg/ml. The in vivo activity of daptomycin at two different doses (standard dose [SD-daptomycin], 6 mg/kg of body weight/day intravenously [i.v.]; high dose [HD-daptomycin], 10 mg/kg/day i.v.) was evaluated in a rabbit model of infective endocarditis and compared with that of a standard dose of vancomycin (SD-vancomycin; 1 g i.v. every 12 h) for 2 days. For the MRSE-375 strain, high-dose vancomycin (HD-vancomycin; 1 g i.v. every 6 h) was also studied. For MRSE-375, SD- and HD-daptomycin therapy sterilized significantly more vegetations than SD-vancomycin therapy (9/15 [60%] and 11/15 [73%] vegetations, respectively, versus 3/16 [19%] vegetations; P = 0.02 and P = 0.002, respectively). HD-daptomycin sterilized more vegetations than HD-vancomycin (11/15 [73%] versus 5/15 [33%] vegetations; P = 0.03) and was more effective than SD- and HD-vancomycin in reducing the density of bacteria in valve vegetations (0 log(10) CFU/g vegetation [interquartile range {IQR}, 0 to 1 log(10) CFU/g vegetation] versus 2 log(10) CFU/g vegetation [IQR, 2 to 2 log(10) CFU/g vegetation] and 2 log(10) CFU/g vegetation [IQR, 0 to 2.8 log(10) CFU/g vegetation]; P = 0.002 and P = 0.01, respectively). For the NRS6 strain, SD- and HD-daptomycin were significantly more effective than vancomycin in reducing the density of bacteria in valve vegetations (3.7 log(10) CFU/g vegetation [IQR, 2 to 6 log(10) CFU/g vegetation] versus 7.1 log(10) CFU/g vegetation [IQR, 5.2 to 8.5 log(10) CFU/g vegetation]; P = 0.02). In all treatment arms, isolates recovered from vegetations remained susceptible to daptomycin and vancomycin and had the same MICs. In conclusion, daptomycin at doses of 6 mg/kg/day or 10 mg/kg/day is more effective than vancomycin for the treatment of experimental endocarditis due to MRSE and GISE.

From 27 April to 16 December 2009, we analyzed the hemagglutinin gene sequence of 2009 pandemic influenza A (H1N1) virus in 189 respiratory specimens. We only found the D225G mutation in 3 severe cases. However, it was not found in samples from other cases with or without clinical criteria of severity. The biologic significance of this mutation remains still unclear., (Copyright 2010 Elsevier Inc. All rights reserved.)

We report the first strain of 2009 pandemic influenza A (H1N1) virus with V27A mutation in addition to S31N substitution in M2 gen. It was isolated from adamantane non-treated child., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability., Methodology/principal Findings: Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 microg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant., Conclusions/significance: In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.

This study evaluated the activity of daptomycin combined with either gentamicin or rifampin against three methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates in vitro and one isolate in vivo against a representative strain (MRSA-572). Time-kill experiments showed that daptomycin was bactericidal against these strains at concentrations over the MIC. Daptomycin at sub-MIC concentrations plus gentamicin at 1x and 2x the MIC yielded synergy, while the addition of rifampin at 2 to 4 microg/ml resulted in indifference (two strains) or antagonism (one strain). The in vivo activity of daptomycin (6 mg/kg of body weight once a day) was evaluated +/- gentamicin (1 mg/kg intravenously [i.v.] every 8 h [q8h]) or rifampin (300 mg i.v. q8h) in a rabbit model of infective endocarditis by simulating human pharmacokinetics. Daptomycin plus gentamicin (median, 0 [interquartile range, 0 to 2] log10 CFU/g vegetation) was as effective as daptomycin alone (0 [0 to 2] log10 CFU/g vegetation) in reducing the density of bacteria in valve vegetations (P = 0.83), and both were more effective than daptomycin plus rifampin (3 [2 to 3.5] log10 CFU/g vegetation; P < 0.05) for the strain studied. In addition, daptomycin sterilized a ratio of vegetations that was similar to that of daptomycin plus gentamicin (10/15 [67%] versus 9/15 [60%]; P = 0.7), and both regimens did so more than daptomycin plus rifampin (3/15 [20%]; P = 0.01 and P = 0.02, respectively). No statistical difference was noted between daptomycin plus gentamicin and daptomycin alone for MRSA treatment. In the combination arm, all isolates from vegetations remained susceptible to daptomycin, gentamicin, and rifampin. Sixty-one percent of the isolates (8/13) acquired resistance to rifampin during monotherapy. In the daptomycin arm, resistance was detected in only one case, in which the daptomycin MIC rose to 2 microg/ml among the recovered bacteria. In conclusion, the addition of gentamicin or rifampin does not enhance the effectiveness of daptomycin in the treatment of experimental endocarditis due to MRSA.

Introduction: The objective of this investigation was to identify the mechanism of decreased susceptibility to gentamicin in a Salmonella clinical isolate, leading to the detection of a aminoglycoside acetyltransferase gene found in a class 1 integron., Methods: A multidrug-resistant Salmonella strain was recovered from feces of a traveler to Egypt. The antimicrobial susceptibility test to 12 antimicrobial agents was performed with the Kirby-Bauer method. The presence of class 1 integron was determined by PCR. The amplified product was recovered and sequenced in order to establish the genes carried. In addition, susceptibility to gentamicin C1a, gentamicin C1, sisomicin, neomycin, dibekacin, kanamycin, tobramycin, amikacin, netilmicin, apramycin, dactimicin, spectinomycin, streptomycin, lividomycin and butirosin, was established. The Champion pET101 Directional TOPO Expression Kit was used to clone and express the aac(3)-I gene., Results: The isolate was identified as Salmonella enterica serovar Haifa, showing resistance to nalidixic acid, tetracycline and decreased susceptibility to gentamicin. One integron with a size circa 1,500 bp, encoding an aac(3)-Id plus aadA7 genes was observed. The analysis of the susceptibility to different aminoglycosides in the E. coli TOP10F' transformed with the vector carrying aac(3)-Id gene showed resistance to gentamicin C1a, gentamicin C1, and dactimicin, in accordance with the presence of this enzyme but, was susceptible to sisomicin. The homology of the amino acid and nucleotide sequences with the AAC(3)-Id enzyme was of 100%., Conclusion: The presence of the AAC(3)-Id enzyme was described for the first time in a S. Haifa.

The incidence of Pneumocystis jirovecii pneumonia (PCP) in HIV-infected patients has decreased thanks to sulfa prophylaxis and combined antiretroviral therapy. The influence of P. jirovecii dihydropteroate synthase (DHPS) gene mutations on survival is controversial and has not been reported in Spain. This prospective multicenter study enrolled 207 HIV-infected patients with PCP from 2000 to 2004. Molecular genotyping was performed on stored specimens. Risk factors for intensive care unit (ICU) admission and mortality were identified using a logistic regression model. Seven patients (3.7%; 95% confidence interval [CI], 1.5-7.5%) had DHPS mutations. Overall mortality was 15% (95% CI, 10-21%), rising to 80% (95% CI, 61-92%) in patients requiring mechanical ventilation. None of the patients with DHPS mutants died, nor did they need ICU admission or mechanical ventilation. PaO(2) <60 mm Hg at admission was a predictor of ICU admission (P = 0.01), and previous antiretroviral therapy predicted non-ICU admission (P = 0.009). PaO(2) <60 mm Hg at admission and ICU admission during the 1st week were predictors of mortality (P = 0.03 and P < 0.001, respectively). The prevalence of DHPS mutants in Spain is low and is not associated with a worse outcome. Severe respiratory failure at admission is the strongest predictor of PCP outcome.

Daptomycin is a lipopeptide antibiotic with potent in vitro activity against gram-positive cocci, including Staphylococcus aureus. This study evaluated the in vitro and in vivo efficacies of daptomycin against two clinical isolates: methicillin-resistant S. aureus (MRSA) 277 (vancomycin MIC, 2 microg/ml) and glycopeptide-intermediate S. aureus (GISA) ATCC 700788 (vancomycin MIC, 8 microg/ml). Time-kill experiments demonstrated that daptomycin was bactericidal in vitro against these two strains. The in vivo activity of daptomycin (6 mg/kg of body weight every 24 h) was evaluated by using a rabbit model of infective endocarditis and was compared with the activities of a high-dose (HD) vancomycin regimen (1 g intravenously every 6 h), the recommended dose (RD) of vancomycin regimen (1 g intravenously every 12 h) for 48 h, and no treatment (as a control). Daptomycin was significantly more effective than the vancomycin RD in reducing the density of bacteria in the vegetations for the MRSA strains (0 [interquartile range, 0 to 1.5] versus 2 [interquartile range, 0 to 5.6] log CFU/g vegetation; P = 0.02) and GISA strains (2 [interquartile range, 0 to 2] versus 6.6 [interquartile range, 2.0 to 6.9] log CFU/g vegetation; P < 0.01) studied. In addition, daptomycin sterilized more MRSA vegetations than the vancomycin RD (13/18 [72%] versus 7/20 [35%]; P = 0.02) and sterilized more GISA vegetations than either vancomycin regimen (12/19 [63%] versus 4/20 [20%]; P < 0.01). No statistically significant difference between the vancomycin HD and the vancomycin RD for MRSA treatment was noted. These results support the use of daptomycin for the treatment of aortic valve endocarditis caused by GISA and MRSA.

Fifty million people are estimated to travel from industrial countries to the tropics annually. In spite of exhaustive studies and widely different diagnosis among returned patients, some cases of febrile illnesses remain without an etiological diagnosis, suggesting that these cases could be due to viral respiratory tract infections. From August 2005 to October 2006, 118 febrile patients without a specific diagnosis in their first visit at the Center for International Health of the Hospital Clínic of Barcelona were included. In all of them, in order to study respiratory viruses, a nasopharyngeal swab was collected. Clinical and radiological features and epidemiological data, as well as other samples for microbiologic studies, were also collected during consultation. Based on the physician's judgment at the time of consultation, patients were classified into four groups: respiratory symptoms (62%), febrile syndrome with nonspecific symptoms (24%), digestive symptoms (10%), and patients presenting both respiratory and digestive symptoms (4%). A pathogen microorganism was detected in 61 patients (52%). Respiratory viruses were detected in 44 out of 118 (37%) travelers included in the study, representing 56% of the patients with respiratory symptoms. The most frequently viruses detected were influenza virus (38%), rhinovirus (23%), adenovirus (9%), and respiratory syncytial virus (9%). Respiratory viruses have been shown to play an important role in imported fever. In light of the fact that international tourism is an increasing phenomenon, new strategies to prevent the spread of respiratory viruses should be considered, specially for influenza when a vaccine is available.

Community-acquired pneumonia (CAP) is a serious lower respiratory tract infection associated with significant morbidity and mortality in immunocompromised patients. The present study evaluated the clinical spectrum of CAP in immunocompromised hosts and the role of respiratory viruses, as well as the yield of viral diagnostic methods. Conventional microbiological tests were routinely performed in immunocompromised patients with CAP. Nasopharyngeal swabs were processed for respiratory viruses by indirect immunofluorescence assay, cell culture and PCR. Four groups were defined according to aetiology of CAP, as follows: group 1 (nonviral), group 2 (mixed, nonviral and viral), group 3 (only viral) and group 4 (unknown aetiology). Over a 1-yr period, 92 patients were included. An aetiological diagnosis was achieved in 61 (66%) patients: 38 (41%), group 1; 12 (13%), group 2; and 11 (12%), group 3. The most frequent pathogen detected was Streptococcus pneumoniae (n = 29, 48%), followed by rhinovirus (n = 11, 18%). PCR identified 95% of respiratory viruses. Clinical characteristics could not reliably distinguish among the different aetiological groups. Respiratory viruses represent a substantial part of the aetiologies of community-acquired pneumonia in immunocompromised patients and its routine assessment through PCR in nasopharyngeal swabs should be considered in the clinical care of these patients.

Neonatal meningitis and septicemia caused by Escherichia coli are still major health problems in industrialized countries. Forty-seven E. coli strains causing neonatal sepsis were analyzed. Twenty-two and 25 strains caused early (detected from 0 to 3 days after birth) and late (detected from 4 to 28 days after birth) infections, respectively. Only the ibeA gene was significantly more prevalent in the strains causing early infections.

The aims of the study were to evaluate the incidence and the clinical implications of human herpesvirus (HHV)-7 primary infection in patients undergoing kidney transplantation and the probable interactions between the three beta-herpesviruses (cytomegalovirus [CMV], HHV-6, and HHV-7). Sixty kidney transplant recipients had sequential plasma and whole blood samples collected prior to transplantation and at 7, 14, 21, 28, 45, 60, 75, 90, and 180 days posttransplantation. We used indirect immunofluorescence assays to detect HHV-7 immunoglobulin (Ig) G antibodies in plasma and quantitative real-time polymerase chain reaction to assess CMV, HHV-6 and HHV-7 viral loads. Sixteen out of 60 patients (27%) did not show HHV-7 IgG antibodies prior to transplantation and they were selected for this study. Whereas 3 (18.75%) out of the 16 HHV-7 seronegative patients seroconverted after transplantation, only one patient (6%) had a high HHV-7 viral load from the seventh day posttransplantation in consecutive blood samples during follow-up without clinical manifestations. In our study, the incidence of posttransplant HHV-7 primary infection was low and asymptomatic.

The efficacy of telavancin, a novel lipoglycopeptide, was evaluated in experimental endocarditis in rabbits using two clinical isolates of glycopeptide-intermediate Staphylococcus aureus: ATCC 700788 and HIP 5836. Infected rabbits were treated for 2 days with telavancin (10 mg/kg of body weight once daily intravenously) or vancomycin (1 g twice daily intravenously), administered with a computer-controlled infusion pump system simulating human serum kinetics. Vegetations were harvested at 16 h postinoculation in the control group and at the end of treatment in the drug-treated group. For ATCC 700788, MICs and minimal bactericidal concentrations (MBCs), respectively, were 1 mg/liter and 4 mg/liter for telavancin and 8 mg/liter and 128 mg/liter for vancomycin. For HIP 5836, MICs and MBCs, respectively, were 4 mg/liter and 8 mg/liter for telavancin and 8 mg/liter and 128 mg/liter for vancomycin. Peak and trough levels were 90 microg/ml and 6 microg/ml, respectively, for telavancin and 46 microg/ml and 6 microg/ml, respectively, for vancomycin. In glycopeptide-intermediate S. aureus ATCC 700788, telavancin sterilized 6 of 16 vegetations (37%), whereas vancomycin sterilized 4 of 20 (20%) (P = 0.29) compared with 0 of 17 in the control group. In HIP 5836 experiments, telavancin and vancomycin sterilized 5 of 16 (31%) and 1 of 15 (7%) vegetations (P = 0.17), respectively, compared with none in the control group. Telavancin reduced vegetation titers by 2.0 and 2.3 logs greater than vancomycin for the ATCC 700788 (4.6 [2.0 to 5.8] versus 6.6 [2.0 to 6.9] log CFU/g vegetation; P = 0.05) and HIP 5836 (4.4 [2.0 to 7.4] versus 6.7 [4.5 to 8.7] log CFU/g vegetation; P = 0.09) strains, respectively; these differences did not reach statistical significance. All isolates from vegetations remained susceptible to telavancin after therapy. The results suggest that telavancin may be an effective treatment for endocarditis caused by glycopeptide-intermediate S. aureus.

A polymerase chain reaction (PCR)-based test for Pneumocystis jiroveci (formerly Pneumocystis carinii f. sp. hominis) might be an alternative to histologic diagnoses of P. jiroveci pneumonia (PCP). However, previously developed nested PCR methods tend to have low specificities (high false-positive rates). In this study, nested and quantitative real-time PCR methods for the amplification of the P. jiroveci DHPS (dihydropteroate synthase) gene were evaluated in a variety of stored clinical samples from Spain, South Africa, and Brazil. The sensitivities of both assays were high, ranging from 62.5% to 100% depending on the type of specimen. In a subset of 71 microscopically confirmed PCP cases and 70 negative cases, the sensitivities and specificities were 94% and 81% for nested PCR and 94% and 96% for real-time PCR, respectively. Real-time PCR has a statistically significantly better specificity than nested PCR (P = .015) and is likely to generate fewer false positives.

In patients with Escherichia coli bacteremia, data on the relationship of phylogenetic background, biofilm production, and degree of bacteremia with clinical variables and prognosis are scarce. During a 1-year period, all adults with bacteremia due to Escherichia coli diagnosed at a university center were enrolled. Determination of phylogenetic background, biofilm production, and genotyping was performed with all strains, and the time to positivity of blood culture vials was recorded. A total of 185 episodes of diverse-source E. coli bacteremia was analyzed. Strains of phylogroup D were predominant (52%). Phylogroup A isolates were associated with pneumonia and prior antibiotic intake, B1 with an abdominal source of infection, B2 with the absence of urological abnormalities, and D with urological abnormalities and age below 65 years. Resistance to antibiotics and no biofilm production were concentrated in phylogroup A strains. Biofilm production was not associated with any clinical variable. An immunocompromising condition (odds ratio [OR] = 5.01, 95% confidence interval [CI] = 1.4 to 17.9), peritonitis (OR = 17, 95% CI = 3.32 to 87), pneumonia (OR = 9.97, 95% CI = 1.96 to 50.6), and

Escherichia coli is the most common microorganism causing urinary tract infections. Quinolone-resistant E. coli strains have fewer virulence factors than quinolone-susceptible strains. Several urovirulence genes are located in pathogenicity islands (PAIs). We investigated the capacity of quinolones to induce loss of virulence factors such as hemolysin, cytotoxic necrotizing factor 1, P fimbriae, and autotransporter Sat included in PAIs in three uropathogenic E. coli strains. In a multistep selection, all strains lost hemolytic capacity at between 1 and 4 passages when they were incubated with subinhibitory concentrations of ciprofloxacin, showing a partial or total loss of the PAI containing the hly (hemolysin) and cnf-1 (cytotoxic necrotizing factor 1) genes. RecA(-) mutants were obtained from the two E. coli strains with partial or total loss of the PAI. The inactivation of the RecA protein affected only the partial loss of the PAI induced by quinolones. No spontaneous loss of PAIs was observed on incubation in the absence of quinolones in either the wild-type or mutant E. coli strains. Quinolones induce partial or total loss of PAIs in vitro in uropathogenic E. coli by SOS-dependent or -independent pathways, respectively.

Background: The role of viruses in community-acquired pneumonia may have been previously underestimated. We aimed to study the incidence and clinical characteristics of community-acquired pneumonia (CAP) due to respiratory viruses in adults adding PCR to routine conventional laboratory tests., Methods: Consecutive adult patients diagnosed of CAP from January 2003 to March 2004 were included. Conventional tests including cultures of blood, sputum, urine antigen detection of Streptococcus pneumoniae and Legionella pneumophila, and paired serologies were routinely performed. Nasopharyngeal swabs were processed for study of respiratory viruses through antigen detection by indirect immunofluorescence assay, isolation of viruses in cell culture and detection of nucleic acids by two independent multiplex RT-PCR assays. According to the aetiology, patients were categorized in 4 groups: group 1, only virus detected; group 2, only bacteria detected; group 3, viral and bacterial; and group 4, unkown aetiology., Results: Of 340 patients diagnosed with CAP, 198 had nasopharyngeal swabs available and were included in this study. Aetiology was established in 112 (57%) patients: group 1, n=26 (13%); group 2, n=66 (33%); group 3, n=20 (10%). The most common aetiological agent was S. neumoniae (58 patients, 29%), followed by respiratory viruses (46 patients, 23%). Forty-eight respiratory viruses were identified: influenza virus A (n=16), respiratory syncytial virus A (n=5), adenovirus (n=8), parainfluenza viruses (n=5), enteroviruses (n=1), rhinoviruses (n=8) and coronavirus (n=5). There were two patients coinfected by two respiratory viruses. Serology detected 6 viruses, immunofluorescence 8, viral culture 12, and PCR 45. For the viruses that could be diagnosed with conventional methods, the sensitivity and specificity of RT-PCR was 85% and 92%, respectively. The only clinical characteristic that significantly distinguished viral from bacterial aetiology was a lower number of leukocytes (P=0.004)., Conclusion: PCR revealed that viruses represent a common aetiology of CAP. There is an urgent need to reconsider routine laboratory tests for an adequate diagnosis of respiratory viruses, as clinical characteristics are unable to reliably distinguish viral from bacterial aetiology.

The effect of ciprofloxacin and two mar RAB inducers on the susceptibility to beta-lactam antibiotics in two AmpC beta-lactamase semi-constitutive producer Citrobacter freundii clinical isolates (the DM 1 and DM 2 strains) was studied. Possible changes in outer membrane protein expression, permeability to cephaloridine, active efflux, and hydrolytic activity of beta-lactamase-crude extracts were evaluated under the influence of ciprofloxacin, sodium salicylate, and 2,4-dinitrophenol. Results were compared with those of the effect of the same three chemicals on a normally beta-lactamase-inducible wild-type C. freundii strain. The three assayed compounds decreased beta-lactamase hydrolysis on cephaloridine in both the two clinical isolates as well as in the wild-type strain. However, only the DM 1 and DM 2 strains showed increased susceptibility to beta-lactams. Sodium salicylate and 2,4-dinitrophenol, but not ciprofloxacin, reduced the expression of a 45-kDa outer membrane protein in the three studied strains, which was accompanied by a 4- to 20-fold diminution in permeability to cephaloridine. In conclusion, two mar RAB inducers and ciprofloxacin induced the Mar phenotype and repressed AmpC beta-lactamase synthesis in the DM 1 and DM 2 clinical isolates.

The aim of this study was to compare different fluoroquinolones against Campylobacter spp., analysing the molecular mechanisms of resistance. Moxifloxacin exhibited the greatest activity of the quinolones tested, being active against isolates carrying a single mutation in the gyrA gene. High resistance levels to moxifloxacin were related to the presence of a double gyrA mutation.

The prevalence of 31 virulence factors was analyzed among nalidixic acid-susceptible and -resistant Escherichia coli strains from phylogenetic group B2. Hemolysin, cytotoxic necrotizing factor 1, and S and F1C fimbriae genes were less prevalent among nalidixic acid-resistant E. coli strains. Quinolone resistance may be associated with a decrease in the presence of some virulence factors.

Amikacin-resistant Escherichia coli strains are isolated rarely from clinical samples. In the present study, investigation of an amikacin-resistant clinical isolate of E. coli demonstrated the presence of two class 1 integrons carrying the aacA4 gene plus the aacA7 gene, and the dfrA17 gene plus the aadA5 gene, respectively. Resistance to amikacin in this E. coli isolate was related to the presence of both aacA4 and aacA7.

Molecular analysis of 63 Escherichia coli urine isolates showed that pyelonephritis (n=23) and prostatitis (n=17) isolates exhibited more virulence factors (VFs) among the 35 sought than did cystitis isolates (n=23). Several nontraditional VFs--including bmaE (M fimbriae), gafD (G fimbriae), fyuA (yersiniabactin receptor), ireA and iroN (novel siderophore receptors), cvaC (colicin [microcin] V), traT (serum-resistance associated), ibeA (invasion of brain endothelium), ompT (outer membrane protease T), and malX (pathogenicity island marker)--either differentiated significantly between syndromes (despite small numbers of isolates and possible multiple-comparison artifacts) or were broadly prevalent. Thus, interventions that target conserved uro-VFs may be possible, despite the likely existence of syndrome-specific pathogenetic mechanisms and/or host defense systems.

Setting: Bata and Malabo districts, Equatorial Guinea, 1 March 1999 to 28 February 2001., Objective: To study the molecular epidemiology of tuberculosis (TB)., Results: During the study period, 429 patients were diagnosed with TB in the Bata and Malabo districts. A positive culture was obtained in 206 (48%) TB patients, with RFLP analysis being performed in 185 (89.8%). Ninety-two different patterns were identified. Single patterns were found in 71 strains (38.3%) and the remaining 114 strains (61.6%) were classified into 21 clusters (of 2 to 25 patients). In addition, 37 of the typing strains were resistant to one or more anti-tuberculosis drugs, and 30 were included in clusters (81%), with 21 low level isoniazid (MIC < or = 1 microg/ml) resistance strains in the same cluster. Statistical analysis showed that resistance to anti-tuberculosis drugs (OR 3.1; 95% CI 1.2-7.6; P = 0.014), and positive smear results (4+ grade smear) (OR 4.3; 95% CI 1.5-12; P = 0.005), were significantly more frequent among patients with clustered strains. No epidemiological links were related to clustering., Conclusions: The level of clustering (61.6%) observed suggests a high degree of recent transmission and a predominance of determined patterns of Mycobacterium tuberculosis strains among the population of Equatorial Guinea.

The aim of this work was to determine whether diazepam could induce the multiple antibiotic resistance (Mar) phenotype in Klebsiella pneumoniae and Escherichia coli strains. The Mar phenotype is characterized by decreased susceptibility to multiple antibiotics due to the loss of porins and/or increased expression of active efflux systems. The effect of subinhibitory concentrations of diazepam on the susceptibility of different antimicrobial agents, outer-membrane protein expression and norfloxacin intracellular accumulation was studied. The results revealed that diazepam concentrations equal or twice adult dosage induced the same Mar phenotype as two well known E. coli marRAB inducers, sodium salicylate and sodium benzoate. Susceptibility to norfloxacin in a K. pneumoniae clinical isolate and E. coli strain Ag100 decreased due to enhanced active efflux and loss of porin expression. A decreased susceptibility to chloramphenicol, tetracycline, nalidixic acid and beta-lactam antibiotics was also observed. In conclusion, like sodium salicylate or sodium benzoate, diazepam may induce the Mar phenotype.

Conventional antifungal therapy for fungal endocarditis has been associated with a poor cure rate. Therefore, combined medical and surgical therapy has been recommended. However, new potent antifungal agents, such as echinocandins, could increase the medical options and, in some cases, avoid the need for surgery. We report a case of Candida endocarditis treated successfully without valve replacement with intravenous liposomal amphotericin B (total dose, 4 g) and intravenous caspofungin (a 100-mg loading dose followed by 50 mg per day for 8 weeks) as induction therapy and intravenous caspofungin (100 mg 3 times per week for 12 weeks) as maintenance therapy.

The evolution of antimicrobial resistance in Salmonella isolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding beta-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]). Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each). Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene. Further surveillance of the Salmonella spp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.

The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.

Objectives: The aim of this study was to determine the roles of mutations in the gyrA and parC genes and the overexpression of efflux pump(s) as mechanisms of resistance to quinolones. Forty-five Yersinia enterocolitica O:3 clinical isolates (41 nalidixic acid-resistant, three nalidixic acid-susceptible and one nalidixic acid-resistant strain obtained in vitro) were analysed., Results: All the nalidixic acid-resistant strains showed mutations in the gyrA gene and none in the parC gene. The presence of the inhibitor produced decreases in the MIC values of nalidixic acid by two to six serial dilution steps in 37 of the 41 nalidixic acid-resistant strains. Meanwhile, the MIC value of ciprofloxacin was affected in two strains whose values diminished three serial dilution steps. The nalidixic acid-resistant mutant obtained in vitro was also affected by the inhibitor decreasing the MIC value of nalidixic acid three serial dilutions steps whereas the MICs for the nalidixic acid-susceptible strains were not affected., Conclusions: Our results show that the high level of resistance to nalidixic acid is likely due to an overexpression of an efflux pump plus a mutation in the gyrA gene, whereas decreased susceptibility to ciprofloxacin is only associated with the presence of a mutation in the gyrA gene.

The Phoenix system (BD Diagnostic Systems), a rapid ID/AST system, was compared with the MicroScan WalkAway-40 system for accuracy of identification and antimicrobial susceptibility test results. The 327 bacterial isolates, were comprised of 191 Gram-negative bacilli (187 Enterobacteriaceae and 4 Aeromonas spp.) and 136 Gram-positive cocci (27 Staphylococcus aureus, 53 coagulase-negative staphylococci, 45 enterococci and 11 beta haemolytic streptococci). The overall rate of agreement between the two systems for species level identification was 95.8% and 96.3% for Gram-negative bacilli and Gram-positive cocci, respectively. Enterococcus and Streptococcus species both achieved a 100% rate of species level agreement. The genus level agreement was >99% overall. Arbitration of the 8 Gram-negative bacilli disagreements resolved with 7 in agreement with the Phoenix identification. For the 5 Gram-positive cocci disagreements, 3 resolved in agreement with Phoenix. Overall, 3688 antimicrobial/organism combinations were evaluated in both systems. For Gram-negative isolates, the rate of essential agreement for the MICs was 98.5%, while the categorical agreement rate was 95.9%. Arbitration of 13 Gram-negative disagreements resolved with 11 in agreement with the Phoenix system. For Staphylococcus spp. and Enterococcus spp. isolates, the essential agreement rates were 96.4% and 99% respectively. Categorical agreement rates for both genera were 94.7% and 96.1%, respectively. Arbitration of 5 staphylococci disagreements resolved with 2 in agreement with Phoenix system. Our results show that the Phoenix system is a rapid and reliable system for both identification and antimicrobial susceptibility testing of common clinical isolates.

Background and Objective: The use of risk indexes, originally developed in the US for the assessment of SSI risk, is an useful instrument that must be analyzed according to each specific procedure. The addition of other possible SSI risk factors, like the use of perioperative antibiotic prophylaxis, could improve the predictive value of these indexes. The aim of this study was to determine the SSI incidence rate for craniotomy in patients admitted to the Neurosurgical Unit of the Hospital Clinic of Barcelona (Spain), to assess the use of standard NNIS and SENIC indexes, and to assess the possible effect of the addition of a new risk factor (adequate or inadequate use of perioperative antibiotic prophylaxis) to these indexes., Patients and Method: Risk factors for SSI were assessed following common standard definitions and procedures (CDC-NNIS) over a three-year period (1999-2001). NNIS and SENIC risk indexes were calculated. The effect of the addition of a new variable, namely perioperative antibiotic prophylaxis adequate (0 points) or inappropriate/no prophylaxis (1 point) on these indexes (modified indexes NNISa and SENICa) was also assessed. Statistical analysis included both parametric and non-parametric standard tests., Results: The study included a total of 203 patients undergoing a craniotomy procedure (40% of all neurosurgical procedures). The overall SSI incidence rate was 6.8% (14 patients developed SSI). The cut-off point (75 percentile) for the duration of the procedure was 180 minutes instead of the commonly US reported 240 minutes. Patients who develop SSI had a trend towards having shorter operation times. For those patients in the lower risk groups, the SSI incidence rate was: NNIS (0, 1): 6.9%; SENIC (0, 1): 6.2%. If the modified indexes were used, the SSI incidence rate was: NNISa (0, 1): 4.2%; SENICa (0, 1): 4.9%. When NNIS and SENIC indexes, both standard and modified (NNISa and SENICa), were compared, no statistically significant differences between infected and non-infected patients were observed., Conclusions: When applied to a health system other than the US, SENIC and NNIS indexes could be useful if adapted to each specific situation and procedure. The added value of a new risk factor (perioperative antibiotic prophylaxis) on standard NNIS and SENIC indexes shows a slight improvement in their prediction rate for SSI in patients undergoing craniotomy, mainly in those patients at lower risk for developing superficial SSI.

Setting: Five districts in Equatorial Guinea, March 1999 to February 2001., Objectives: To determine tuberculosis drug resistance among new and previously treated cases, the risk factors associated with resistance, and the mutations associated with isoniazid and rifampicin (katG, inhA and rpoB genes) resistance, and to genotype resistant strains., Results: A positive culture identified as Mycobacterium tuberculosis complex was obtained in 240/499 patients. Susceptibility testing was performed in 236 strains. The overall resistance rate in new cases was 16.9% compared to 41.6% in previously treated cases. Isoniazid resistance was the most frequent (respectively 12.5% and 16.6%) in the two groups, while multidrug resistance was observed in 1.7% and 25% of new and previously treated cases, respectively. Female sex was statistically associated with resistance in new cases. Of 41 isoniazid-resistant strains, 33 (80.5%) had mutations in the inhA gene; none had mutations in the katG gene and eight had no mutations in either gene. All strains had low-level isoniazid resistance. Of eight strains resistant to rifampicin, six had mutations in the rpoB gene. Genotyping defined seven clusters., Conclusions: Moderate resistance was found in new cases. Low-level isoniazid resistance predominated among mutations in the inhA gene, with a high percentage of clustering in resistant strains.

Antibiotic resistance in clinical isolates of Salmonella typhimurium has steadily risen in recent years. Some of the resistance genes may be carried into integrons. In this study, integrons, both from 10 epidemiologically related and unrelated S. typhimurium clinical isolates, were characterized, showing that epidemiologically different strains can carry the same integron, and that epidemiologically related strain can carry different integrons. Among the resistance genes detected in this study were genes encoding b-lactamases (bla(oxa-30) in two strains, and bla(pse-1) in five strains, one of which was carrying this cassette in two different integrons); aminoglycoside-modifying enzymes (aadA2 in four strains, one of which was carrying this cassette in two different integrons, and aadA1 in six strains); as well dihydrofolate reductases (dfrAI in three strains).