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Twenty-two carbapenem-resistant enterobacterial isolates were recovered from patients hospitalized between October 2010 and March 2011 at the Royal Hospital of Muscat, Sultanate of Oman. Eleven NDM-1, five OXA-48 and one NDM-1 plus OXA-181 producers of diverse ST types were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were nearly always associated to other resistance determinants, including extend-spectrum ß-lactamases and the ArmA methylase encoding resistance to aminoglycosides. This work highlights the dissemination of NDM-1 and OXA-48-type producers in the Middle East.© 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases 

 

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Hepatitis E virus (HEV) is one of the leading agents of acute hepatitis. This study investigated the prevalence and risk factors of HEV infection in Tunisian adult general population, either in blood donors (n=687) or in patients hospitalised for acute hepatitis (n=202). The mode of transmission differed between these two populations: contact with animals and living in rural habitat were the main risk factors of being in contact with HEV in asymptomatic blood donors, while HEV was contracted through contaminated water in symptomatic cases. HEV seroprevalence in adult blood donors in Tunisia was relatively low (5.4%) and increased with age.© 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases 

 

Sickle cell anemia (SCA) is a hemoglobin disorder, which alters the deformability of erythrocytes due to abnormal polymerization of hemoglobin. Children with SCA have an increased risk of infections with encapsulated bacteria. To guide the antibiotic prophylaxis and vaccinations in SCA children in Gabon, we characterized Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae from children with and without SCA.We performed a cross-sectional study and compared nasal and pharyngeal S. pneumoniae, S. aureus and H. influenzae isolates from SCA children (n = 73) with age-, residence- and sex-matched comparators (n = 143) in a matched-comparison analysis. The resistance pattern and capsular type was identified for each isolate. The total carriage rate for S. pneumoniae, S. aureus and H. influenzae was 13.8%, 46.7% and 12.5%, respectively and did not differ between both groups (p>0.05). The mean number of days under antibiotic treatment in the past year was higher in SCA children than in controls (penicillin: 70.1 vs. 0.1 days, p = 0.00002). The total non-susceptibility rate was 30% for oral and parenteral (meningitis) penicillin in S. pneumoniae, resistance rates were 1.6% for oxacillin in S. aureus and 14.8% for ampicillin in H. influenzae. Susceptibility to antibiotic agents and distribution of capsular types did not differ significantly between both groups.In conclusion, carriage and resistance rates are similar in children with and without SCA. Our data provide the basis to guide empiric therapy of invasive diseases caused by S. pneumoniae, S. aureus and H. influenza in children in Gabon. 

 

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An Australian family was identified through a Public Health follow up on a STEC positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence subtyping, MLVA, MLST and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup, which differed in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters. 

 

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A prospective, multicentre, phase IIIb study with an exploratory, open-label design was conducted to evaluate efficacy and safety of anidulafungin for the treatment of candidaemia/invasive candidiasis (C/IC) in specific ICU patient populations. Adult ICU patients with confirmed C/IC meeting 1 of the following criteria were enrolled: postabdominal surgery, solid tumour, renal/hepatic insufficiency, solid organ transplant, neutropaenia, age 65 years. Patients received anidulafungin (200 mg on day 1, 100 mg/day thereafter) for 10–42 days, optionally followed by oral voriconazole/fluconazole. The primary efficacy endpoint was global (clinical and microbiologic) response at the end of all therapy (EOT). Secondary endpoints included global response at the end of intravenous therapy (EOIVT), at 2 and 6 weeks post-EOT, survival at day 90, and incidence of adverse events (AEs). The primary efficacy analysis was performed in the modified intent-to-treat (MITT) population, excluding unknown/missing responses. The safety and MITT populations consisted of 216 and 170 patients, respectively. The most common pathogens were Candida albicans (55.9%), C. glabrata (14.7%), and C. parapsilosis (10.0%). Global success was 69.5% (107/154; 95% CI, 61.6–76.6) at EOT, 70.7% (111/157) at EOIVT, 60.2% (77/128) at 2 weeks post-EOT, and 50.5% (55/109) at 6 weeks post-EOT. When unknown/missing responses were included as failures, the respective success rates were 62.9%, 65.3%, 45.3%, and 32.4%. Survival at day 90 was 53.8%. Treatment-related AEs occurred in 33/216 (15.3%) of patients, four (1.9%) of whom had serious AEs. Anidulafungin was effective, safe, and well tolerated for the treatment of C/IC in selected groups of ICU patients. 

 

For patients hospitalized with pneumonia, guidelines provide empiric antibiotic recommendations and some studies suggest that macrolide/ß-lactam combinations are preferable. We hypothesized that guideline-concordant regimens, particularly macrolide/ß-lactams, would reduce mortality and ICU admissions. All patients hospitalized with pneumonia in Edmonton, Alberta, Canada were managed according to a clinical pathway and enrolled in a population-based registry. Clinical data, Pneumonia Severity Index and treatments were collected. Guideline-concordant regimens were macrolides/ß-lactams or respiratory fluoroquinolone monotherapy. Main outcome was in-hospital mortality. The study included 3203 patients and most had severe pneumonia (63% PSI Class IV-V). 321 (10.0%) patients died, 306 (9.6%) were admitted to ICU and 570 (17.8%) achieved the composite of death or ICU admission. Most (n=2506) patients received guideline-concordant antibiotics. Receipt of guideline-concordant antibiotics was not associated with a reduction in mortality alone (231[9.2%] vs 90[12.9%], adjusted odds ratio (aOR) 0.82, 95%CI 0.61-1.09, p=0.16), but was associated with decreased death or ICU admission (14.7% vs 29.0%, aOR 0.44, 95% CI 0.36-0.54, p<0.0001). Within guideline-concordant subgroups, there was no difference in mortality with macrolide/ß-lactams compared with respiratory fluoroquinolone monotherapy (22[8.3%] vs 209 [9.3%], aOR 1.09, 95% CI 0.66-1.81 p=0.73) but macrolide/ß-lactams were associated with increased odds of death or ICU admission (17.4% vs 14.4%, aOR, 1.58, 95% CI 1.09-2.27, p=0.01). In conclusion, guideline-concordant antibiotics were not associated with decreased mortality for patients hospitalized with pneumonia, but were associated with decreases in the composite endpoint of death or ICU admission. Our findings do not support any clinical advantage to macrolide/ß-lactam compared to respiratory fluoroquinolone monotherapy. 

 

Recent studies of molecular and genomic data from the parasitic lice of birds and mammals, as well as their mutualistic endosymbiotic bacteria, are changing the phylogenetic relationships and taxonomy of these organisms. Phylogenetic studies of lice suggest that vertebrate parasitism arose multiple times from free-living book and bark lice. Molecular clocks show that the major families of lice arose in the late Mesozoic and radiated in the early Cenozoic following the radiation of mammals and birds. The recent release of the human louse genome has provided new opportunities for research. The genome is being used to find new genetic markers for phylogenetics and population genetics, to understand the complex evolutionary relationships of mitochondrial genes, and to study genome evolution. Genomes are informing us not only about lice, but also about their obligate endosymbiotic bacteria. Contrary to lice and their hosts, lice and their endosymbionts do not share common evolutionary histories, suggesting that endosymbionts are either replaced over time or that there are multiple independent origins of symbiosis in lice. Molecular phylogenetics and whole genome sequencing has recently provided the first insight into the phylogenetic placement and metabolic characteristics of these distantly related bacteria. Comparative genomics between distantly related louse symbionts can provide insights into conserved metabolic functions and can help to explain how distantly related species are fulfilling their role as mutualistic symbionts. In lice and their endosymbionts, molecular data and genome sequencing is driving our understanding of their evolutionary relationships and classification and will for the foreseeable future. 

 

Hand, foot and mouth disease (HFMD) and herpangina (HA) are frequently caused by several distinct serotypes belonging to the human enterovirus A species (HEVA). Enterovirus 71 is considered as a significant public health threat because of rare but fatal neurologic complications. A sentinel surveillance system involving paediatricians from Clermont-Ferrand (France) was set up to determine the clinical and epidemiologic characteristics of HFMD/HA associated with EV infections.A standardised report form was used to collect demographic and clinical data. Throat or buccal specimens were obtained prospectively and tested for the presence of enteroviruses. The frequency of HEVA serotypes was determined by genotyping. Phylogenetic relationships were analysed to identify potential new virus variants.From April 1st to December 31st 2010, a total of 222 children were enrolled. HA was the predominant clinical presentation (63.8%) and was frequently associated with clinical signs of HFMD (48%). An EV infection was diagnosed in 143 (64.4%) patients and serotype identification was achieved in 141/143 (98.6%). The predominant EV serotypes were coxsackievirus A10 (39.9%) and A6 (28%), followed by coxsackievirus A16 (17.5%) and enterovirus 71 (6.3%). Fever was observed in 115 (80.4%) children. No patient had neurological complications. Coxsackievirus A10 and A6 strains involved in the outbreak were consistently genetically related with those detected earlier in Finland and constituted distinct European lineages.Although several EV serotypes have been involved in HFMD/HA cases, the outbreak described in this population survey was caused by CV-A6 and CV-A10, the third dual outbreak in Europe in the last three years. 

 

Drawing its etymology from the Latin pestis, curse, the plague, over the last centuries, was more dreaded by mankind than any other epidemic. The Apocalypse had recognised the plague as the archetypal divine curse, the power to kill over a fourth of the earth. The plague is thus a particular topic of study insofar as it is one of the rare epidemics that had recurrent major consequences on demography and human societies. Its highly transmissible feature, the brutality of its action, its high pathogenicity marked by a strong lethality and a great swiftness, the complete absence of therapeutic before the 20th century, conferred to it a sinister specificity. Generating series of severe demographic crisis, rather well-known in the Western world, it has necessarily influenced the evolution of the societies, at both biological and cultural level. 

 

Medical residents are particularly exposed to the risk of occupational infection. We aimed to determine the vaccination coverage in residents with an anonymous self-reporting electronic questionnaire. A total of 250 residents entered this survey. Vaccination rates were particularly high for mandatory vaccinations (diphtheria, tetanus, poliomyelitis, hepatitis B virus and tuberculosis). Regarding recommended vaccinations (influenza 45.6%, pertussis 65.2%, measles 62.8%, varicella 62.8%), rates were insufficient to prevent hospital epidemics, but higher than those reported in other healthcare workers. Further immunization programmes should target residents, and not only senior healthcare workers, with a critical role for occupational medicine departments. 

 

Virulence factors are thought to be responsible for the virulence capacity of pathogenic bacteria. However recently, epidemic bacteria were found to contain significantly fewer virulence factors than non epidemic species and some of the most dangerous epidemic bacteria, such as Mycobacteria spp., or Rickettsia spp. are reduced and contain hundreds of degraded genes. Epidemic bacteria are actually highly specialized species, characterized by allopatric speciation, that after adapting to their hosts, attempt to maintain a balance between gene gain and gene loss favouring gene loss, finally leading to a genome reduction. Recent comparative genomic studies have demonstrated that the specialization of bacteria to eukaryotic cells is associated with massive gene loss. Furthermore, the 12 deadliest epidemic species to mankind have significantly smaller genomes with less ORFs than less dangerous species. Epidemic species mostly loose genes related to metabolic activity, the production of energy, cell motility and transcription. Epidemic bacteria also possess a damaged recombination and repair system and significantly more toxins than closely related non-pathogenic or non-epidemic species, and more toxin-antitoxin modules. Epidemic bacteria are therefore highly specialized species, adapted to their hosts, characterized by an excessive genome reduction. Except from toxins and toxin-antitoxin modules that have a direct and measurable effect, other virulence factors are factors associated with fitness in experimental models. Epidemic species are defined by a virulent genomic repertoire including both present and absent genes. 

 

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Mycobacterium tuberculosis is assumed to remain in a quiescent state during latent infection, being unable to grow in culture.The aim of this study was to evaluate the detection of viable but non-cultivable bacilli with metabolic activity in human clinical samples using a procedure that is independent of the immunological status of the patient.The study was performed on 66 human clinical samples, from patients subjected to routine diagnosis to rule out a mycobacterial infection. Specimens from pulmonary and extra-pulmonary origins were verified to contain human DNA, prior to testing for Mycobacterium tuberculosis DNA, rRNA and transient RNA by real-time quantitative PCR. Clinical records of 55 patients were also reviewed.We were able to detect viable but non-cultivable bacilli with a metabolic activity in both pulmonary and extra-pulmonary samples. Mycobacterium tuberculosis RNA was detected in the majority of culture-positive samples whereas it was detected in one third of culture-negative samples, 20% of them showed metabolic activity.The amplifications of the ftsZ gene and particularly of the main promoter of the ribosomal operon rrnA, namely PCL1, seem to be good targets to detect active bacilli putatively involved in latent infection. Moreover, this last target would give information on the basal metabolic activity of the bacilli detected. 

 

Several of the infectious diseases associated with human lice are life-threatening, including epidemic typhus, relapsing fever, and trench fever, which are caused by Rickettsia prowazekii, Borrelia recurrentis, and Bartonella quintana, respectively. Although these diseases have been known for several centuries, they remain a major public health concern in populations living in poor hygienic conditions due to war, social disruption, severe poverty, or gaps in public health management. Poor hygienic conditions favor a higher prevalence of body lice, which are the main vectors for these diseases. Trench fever has been reported in both developing and developed countries in populations living in poor conditions, such as homeless individuals. In contrast, outbreaks of epidemic typhus and epidemic relapsing fever have occurred in jails and refugee camps in developing countries. However, reports of a significantly high seroprevalence for epidemic typhus and epidemic relapsing fever in the homeless populations of developed countries suggest that these populations remain at a high risk for outbreaks of these diseases. Additionally, experimental laboratory studies have demonstrated that the body louse can transmit other emerging or reemerging pathogens, such as Acinetobacter baumannii and Yersinia pestis. Therefore, a strict survey of louse-borne diseases and the implementation of efficient delousing strategies in these populations should be public health priorities. 

 

The kinetics of serum (13)-ß-D-glucan (BG) following the diagnosis of invasive fungal disease and administration of antifungal therapy are poorly characterized. It is unknown whether early BG changes have prognostic implications. We assessed the post-diagnostic kinetics of BG in patients with an initial serum BG 80 pg/mL and at least one additional post-diagnostic BG value in the setting of invasive aspergillosis (IA, n=69), invasive candidiasis (IC, n=40), or Pneumocystis jirovecii pneumonia (PCP, n=18), treated with antifungal therapy. Clinical failure of antifungal therapy and mortality were assessed at 6 and 12 weeks, and Cox modeling was used to assess the hazard of initial BG and change in BG at 1 or 2 weeks for these outcomes. In patients with 2 BG values, median initial BG was >500 pg/mL (IQR (interquartile range) 168, >500; range 80, >500) in IA, 136 pg/mL (IQR 88, >500; range 31, >500) in IC, and >500 pg/mL (IQR 235, >500; range 86, >500) in PCP. In patients with 2 BG values through one week after diagnosis, overall one-week decline in BG was 0 pg/mL (IQR 0, 53) in IA, 0 (IQR -65, 12) in IC, and 17 (IQR 0, 82) in PCP. Most patients with BG values through 6 and 12 weeks had persistent levels >80 pg/mL. Initial BG and the early trajectory of BG were not predictive of 6 or 12-week clinical failure or mortality. While BG eventually declines in patients with IA, IC, and PCP, it lacks prognostic value within a clinically meaningful time frame. 

 

The impact of bloodstream infection (BSI) on admission to the hospital on the outcome of patients with Community-Acquired Sepsis (CAS) admitted in Intensive Care Units (ICU) is largely unknown. We selected 803 adult patients consecutively admitted with CAS to one of 17 Portuguese ICU, in whom blood cultures were collected before initiation of antibiotic therapy during a 12-month period. A bloodstream infection (BSI) was identified on hospital admission in 160 (19.9%) patients. Those with and without BSI had similar mean Simplified Acute Physiology Score (SAPS) II and age. The presence of BSI was independently associated with mortality in ICU [Adjusted Odds Ratio (AOR)=1.86; 95% confidence interval (CI): 1.20–2.89; p=0.005]. On the fourth ICU day, BSI patients were found to be significantly more dependent of vasopressor (p=0.002) but not of ventilatory support. Cumulative ICU mortality was significantly higher in BSI patients from the ninth day onwards. A seasonal variation of BSI isolates was noted: Gram-negative BSI were more common in the summer, whilst in the winter, Gram-positive were more frequent (p=0.024), without mortality differences. 

 

The Staphylococcus aureus sequence type (ST) 398 commonly found as a colonization strain in pig-farming is emerging more and more as the cause of human infections. In this study we analyzed the ST398 of porcine and human origins on genetic, proteinic, and immunogenic levels. Although the genetic analysis of the genes encoding the major virulence factors revealed the presence of the same genes in all strains studied, the results demonstrate spa type crossing alterations in the adhesion abilities in addition to a strongly enhanced lysis activity directly linked to an impaired clearance due to polymorphonuclear leukocytes (PMN). This change in virulence pattern indicate a high heterogenicity in the ST398 pool which is not based on a different genetic set-up but probably on variations in the genetic regulation systems. These modifications which are tightly connected to pathogenicity cannot be detected by conventional diagnostic methods. 

 

A significant proportion of women develop a recurrence following an initial urinary tract infection (UTI). In women with recurrent UTI (rUTI), the predictive value of asymptomatic bacteriuria (ASB) for the development of a subsequent UTI has not yet been established and it is not known whether information from an asymptomatic sample is useful in guiding antimicrobial therapy. To address these questions, we used data that originated from the Non-antibiotic prophylaxis for recurrent urinary tract infections’ (NAPRUTI) study: two randomized controlled trials on prevention of rUTI in non-hospitalized pre- and postmenopausal women (n=445). During 15 months of follow-up, no difference was observed in the time to a subsequent UTI between women with and without ASB at baseline (hazard ratio: 1.07, 95% confidence interval: 0.80 – 1.42). The antimicrobial susceptibility and pulsed-field gel-electrophoresis (PFGE) pattern of 50 Escherichia coli strains causing a UTI were compared with that of the ASB strain isolated one month previously. The predictive values of the susceptibility pattern of the ASB strain, based on resistance prevalence at baseline, were 76%, except in the case of nitrofurantoin- and amoxicillin-clavulanic acid-resistance. Asymptomatic and symptomatic isolates had similar PFGE patterns in 70% (35/50) of the patients.In the present study among women with rUTI receiving prophylaxis, ASB was not predictive for the development of a UTI. However, the susceptibility pattern of E. coli strains isolated in the month prior to a symptomatic E. coli UTI can be used to make informed choices for empirical antibiotic treatment in this patient population. 

 

With plague being not only a subject of interest for historians, but still a disease of public health concern in several countries mainly in Africa, hopes were put that the analyses of the Yersinia pestis genomes would put this deadly epidemic pathogen down. Genomics revealed that Y. pestis isolates evolved from Yersinia pseudotuberculosis in Central Asia some millennia ago after the acquisition of two Y. pestis-specific plasmids balanced genomic reduction parallel to the expansion of insertion sequences illustrating the modern concept that, except for the acquisition of plasmid-borne toxin-encoding genes, increased virulence of Y. pestis resulted from gene loss rather that gene acquisition. Telluric persistence of Y. pestis reminds of this close relationship and matters in terms of plague epidemiology. While biotype Orientalis isolates spread worldwide, the Antiqua and Medievalis ones had more limited expansion. In addition to animal ectoparasites, human ectoparasites such as the body louse may have participated to this expansion and to devastating historical epidemics. The recent analysis of a Black Death genome indicated it was more closely related to Orientalis branch than to the Medievalis branch. Modern Y. pestis isolates grossly exhibit the same gene content but still undergo microevolution in geographically limited areas by differing in the genome architecture due to inversions near insertion sequences and the stabilisation of the YpfPhi prophage in Orientalis biotype isolates. Genomics provided several new molecular tools for the genotyping and phylogeographical tracing of isolates and description of plague foci. However, genomics and post-genomics did not yet bring new tools for the prevention, diagnosis and management of plague patients and plague epidemics still raging in some sub-saharian countries. 

 

Studies of the type 3 secretion system (T3SS) in Pseudomonas aeruginosa isolates from chronically-infected older children and adults with cystic fibrosis (CF) show a predominantly exoS+/exoU- (exoS+) genotype and loss of T3SS effector secretion over time. Relatively little is known about the role of the T3SS in the pathogenesis of early P. aeruginosa infection in the CF airway. In this longitudinal study, 168 P. aeruginosa isolates from 58 children diagnosed with CF following newborn screening and 47 isolates from homes of families with or without children with CF were genotyped by pulsed-field gel electrophoresis (PFGE) and T3SS genotype and phenotype determined using multiplex PCR and western blotting. Associations were sought between T3SS data and clinical variables and comparisons made between T3SS data of clinical and environmental PFGE genotypes. Seventy-seven of the 92 clinical strains were exoS+ (71% secretors [ExoS+]) and 15 were exoU+ (93% secretors [ExoU+]). Initial exoS+ strains were five times more likely to secrete ExoS than subsequent exoS+ strains at first isolation. The proportion of ExoS+ strains declined with increasing age at acquisition. No associations were found between T3SS characteristics and gender, site of isolation, exacerbation, a persistent strain or pulmonary outcomes. Fourteen of the 23 environmental strains were exoS+ (79% ExoS+) and nine were exoU+ (33% ExoU+). The exoU+ environmental strains were significantly less likely to secrete ExoU than clinical strains. This study provides new insight into the T3SS characteristics of P. aeruginosa isolated from the CF airway early in life. 

 

We reported the first identification of Shigella flexneri serotype 1c in China and also the emergence of resistance to ciprofloxacin and third-generation cephalosporins in serotype 1c the first time. Seven isolates circulating in China were divided into three new MLST sequence types and seven PFGE banding patterns, demonstrating the high genetic diversity. The seven isolates showed reduced susceptibility to ciprofloxacin, among which one had ciprofloxacin-resistance and an additional one developed resistance to ciprofloxacin, norfloxacin, cefotaxime and ceftriaxone. 

 

Enterovirus-positive samples diagnosed in Marseille (January 2009-September 2011) were screened for EV71 by real-time RT-PCR. EV71 was detected in three children below the age of two with no history of overseas travel; two of these cases were associated with severe clinical presentation. Viruses demonstrated genetic similarity with other European genogroup C2 strains. Strain MRS/09/3663 complete sequencing revealed 97.6% identity across the entire genome with a 2008 Singapore isolate, without signs of possible recombination events. To our knowledge, this is the first detection of EV71 infection in Marseille, France, that confirms the current circulation of EV71 in France. 

 

Multidrug-resistant Salmonella infection is a global problem and carbapenems may represent as the last therapeutic choice. We report a case of infection caused by ceftriaxone- and ciprofloxacin-resistant Salmonella enterica serotype Typhimurium. A blaCMY-2-containing Tn6092, located on a self-transferable IncI1 plasmid, was found in all isolates derived from the patient. While on ertapenem treatment, the strain developed carbapenem resistance. Apart from the OmpD deficiency found in all isolates, the strain further developed OmpC deficiency through a single gene mutation and became carbapenem-resistant. Salmonella appears very plastic in developing antimicrobial resistance. Caution must be taken by physicians when treating multidrug-resistant Salmonella infection. 

 

The number of elderly patients in the community with immunosuppressive conditions has increased progressively over recent decades. We sought to determine the incidence, causative organisms and outcome of community-acquired pneumonia (CAP) occurring in immunocompromised older patients. We prospectively compared cases of CAP in immunocompromised and non-immunocompromised patients admitted to five public hospitals in three Spanish regions. Of 320 cases studied, 115 (36%) occurred in immunocompromised patients, including: solid or hematological malignancy (97), corticosteroids or other immunosuppressive drugs (44), solid organ or stem cell transplant (5), and other conditions (8). The etiology was established in 44% of immunocompromised patients vs. 32% of non-immunocompromised patients (p=0.03). Streptococcus pneumoniae was the most common causative organism in both groups (29% vs. 21%; p=0.08), followed by Legionella pneumophila (3% vs. 6%; p= 0.01). Gram-negative bacilli were more frequent among immunocompromised patients (5% vs. 0.5%; p<0.01), particularly Pseudomonas aeruginosa (3% vs. 0%; p=0.04). Nocardiosis was only observed in immunocompromised patients (2 cases). Bacteremia occurred similarly in the two groups. No significant differences were found with respect to ICU admission (8%, in both groups) or the length of stay (12.5 vs. 10.4 days). The early (<48 hours) (3.5 vs. 0.5%; p=0.04) and overall case-fatality rates (12% vs. 3%; p<0.01) were higher in immunocompromised patients. In conclusion, a substantial number of older patients hospitalized for CAP are immunocompromised. Although relatively uncommon, CAP due to gram-negative bacilli, including P. aeruginosa, is more frequent among these patients. CAP occurring in immunocompromised patients causes significant morbidity and mortality. 

 

Limited data exist on Candida endocarditis (CE) outcome in the era of new antifungals. Since early diagnosis of CE remains difficult, non-culture based tools need to be evaluated.Through the French prospective MYCENDO study (2005-2007), the overall characteristics and risk factors for death from CE were analyzed. The contribution of antigen detection (mannan/anti-mannan and (1,3)-ß-D-glucans) and molecular tools was evaluated. Among 30 CE cases, 19 were due to non-albicans species. Sixteen patients (53%) had a predisposing cardiac disease, being a valvular prosthesis in 10 (33%). Nine patients (30%) were intravenous drug users; none of them had right-sided CE. Among the 21 patients who were not IV drug users, 18 (86%) had healthcare-associated CE. Initial therapy consisted in a combination of antifungals in 12/30 patients (40%). Thirteen patients (43%) underwent valve replacement. Median follow-up was 1 year after discharge from hospital (range, 5 months-4 years) and hospital mortality was 37%. On univariate analysis, patients 60 years had a higher mortality risk (OR [95% CI] = 11 [1.2-103.9]; P=0.024) while intravenous drug use was associated with a lower risk of death (0.12 [0.02-0.7]; P=0.03). Among 18 patients screened for both serum mannan/antimannan antibodies and (1,3)-ß-D-glucans, all had at least one of either test positive at CE diagnosis. Real-time PCR was performed on blood (SeptiFast®) in 12/18 and confirmed blood culture results. In conclusion, CE prognosis remains poor with a better outcome among younger patients and intravenous drug users. Detection of serum antigens and molecular tools may contribute to earlier CE diagnosis. 

 

Plasmid-mediated AmpC ß-lactamase-producing E. coli (AmpC-E) bacteraemia was characterised by comparison with bacteraemia caused by extended-spectrum ß-lactamase-producing E. coli (ESBL-E) and non-resistant E. coli (NR-E) in the era of the worldwide spread of the CTX-M-15 producing O25b-ST131-B2 clone. Of 706 bloodstream E. coli isolates collected between 2005 and 2010 in 3 Japanese university hospitals, 111 ESBL screening-positive isolates were analysed for AmpC and ESBL genes by PCR. A case-control study was performed in which the cases consisted of all the patients with AmpC-E bacteraemia. Phylogenetic groups, sequence types, and O25b serotype were determined. Twenty-seven AmpC-E isolates (26 of which were of the CMY-2 type) were identified, and 54 ESBL-E and 54 NR-E isolates were selected for the controls. Nineteen AmpC-E isolates were also positive for ESBL. The CTX-M-14 was the most prevalent ESBL type, both in the AmpC-E and ESBL-E isolates. The O25b-ST131-B2 clone was the most prevalent of the ESBL-E (26%) and the second most prevalent of NR-E (13%), but only one O25b-ST131-B2 clone was found among the AmpC-E isolates. Twenty-three different sequence types were identified among the AmpC-E isolates. When compared to bacteraemia with ESBL-E, previous isolation of multidrug-resistant bacteria and intravascular catheterisation were independently associated with a lower risk for AmpC-E. When compared to NR-E bacteraemia, prior use of antibiotics was the only significant risk factor for AmpC-E. Unlike the spread of the O25b-ST131-B2 clone between ESBL-E and NR-E, the AmpC-E isolates were not dominated by any specific clone. 

 

Two patients with no travel history and sharing the same room were colonized by the same strain of NDM-1-producing Escherichia coli within a geographic area not endemic for this highly multidrug-resistant (HMDR) bacterium. An absence of epidemiological and bacteriological link was demonstrated with a third patient returning from India after surgery and found infected by a NDM-1-producing Citrobacter strain at the same period. Despite extensive investigation, the source of contamination of the two former patients was not elucidated. This case report illustrates the need to investigate rapidly the emergence of HMDR Enterobacteriaceae to stop their dissemination in a nosocomial setting. 

 

Background: Pneumocystis jirovecii pneumonia (PCP) can affect various types of immunocompromised patients. We sought to evaluate the diagnostic accuracy of (13)-ß-D-glucan (BDG) for the diagnosis of PCP.Methods: We did a meta-analysis of relevant studies, identified through PubMed and Scopus. Eligible studies were those that reported BDG diagnostic data in cases with documented PCP and controls with other conditions. Cases of invasive fungal infections or healthy controls were excluded. We performed a bivariate meta-analysis of sensitivity and specificity and constructed a hierarchical summary receiver operating characteristics (HSROC) curve.Results: Fourteen studies were included in the meta-analysis. BDG data were analyzed for 357 PCP cases and 1723 controls. The average (95% confidence interval) sensitivity and specificity of BDG were 94.8% (90.8–97.1%) and 86.3% (81.7–89.9%), respectively. The positive and negative likelihood ratios were 6.9 (5.1–9.3) and 0.06 (0.03–0.11), respectively. The area under the HSROC curve was 0.965 (0.945–0.978).Conclusion: Serum BDG shows excellent sensitivity and very good specificity in the diagnosis of PCP. Still, in clinical practice the test results should be interpreted in the context of the underlying clinical characteristics of the individual patient. 

 

A serological survey was performed to determine the prevalence of antibodies against human bocavirus in Apulian population. Anti-hBoV IgG antibodies were analyzed in 1,206 inhabitants (Age range: 1 month-84 years) using a standardized ELISA test based on the use of recombinant hBoV VP2 virus-like particles. In total, 1,075 (89.1%) of 1,206 participants (mean age 32±24.8 yrs) displayed anti-hBoV-IgG. The seroprevalence increased significantly (p<0.0001) in children from 2-4 yrs (64.2%) to 5-9 yrs (96.4%). A similar trend was observed in both males and females.In conclusion, our results show that hBoV infection is common in population, especially in children. 

 

Although it is known that two-tier serologic testing for Lyme disease may be associated with false positive results on the IgM immunoblot, this problem has never been systematically studied in the clinical practice setting. In a retrospective investigation of patients referred to the private adult practice of an Infectious Diseases physician for possible for Lyme disease, 50 of 182 patients (27.5%, 95% CI: 21.1-34.6) were found to have a false positive IgM immunoblot. 78.0% of these patients had received unnecessary antibiotic therapy. False positive results were not restricted to any single commercial laboratory. Research on alternative testing strategies that eliminate the IgM immunoblot entirely is warranted. 

 

We report a case of viral peritonitis caused by coxsackievirus B1 in a 79-year-old male undergoing continuous ambulatory peritoneal dialysis (CAPD) and review the English literature. Clinicians should be aware of viral peritonitis in patients on CAPD presenting with a viral syndrome and mononuclear PD effluent. Currently, viral diagnostic tests are available to confirm the diagnosis and avoid unnecessary treatment with antibiotics. 

 

The recent finding of a new mecA homologue mecALGA251 with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for MRSA into question. A multiplex PCR was designed to differentiate mecALGA251 from the known mecA together with detection of lukF-PV and the spa gene fragments enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing was validated on a large collection (n=185) of contemporary MRSA and MSSA isolates, including 127 isolates carrying mecALGA251. The mecALGA251 was situated in SCCmec XI elements and sequence variation within a 631 bp fragment of the mecALGA251 gene in 79 isolates indicated a much conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a two-month period, among 203 samples tested, 12 new MRSA cases due to mecALGA251 were identified emphasizing the clinical importance of testing for these new MRSA isolates. 

 

Q fever caused by Coxiella burnetii, may result in abortions in infected animals and pregnant women. However, the role Q fever plays in spontaneous abortions is still unknown. This study examined the association between Q fever serology and abortion in a region where Q fever is endemic. A case-control population-based study was conducted in General Yage Hospital (Burgos area, Spain) between June 2009 to July 2010. A total of 801 samples from 500 pregnant women were tested, of whom 273 had a spontaneous abortion and 227 gave birth. IgG and IgM antibody titres against Q fever were determined in their two phases (I and II) by immunofluorescence assay (IFA). Seropositivity (phase I IgG 1:16 or phase II IgG 1:80) was detected in 88/273 (32.2%) cases and 53/227 controls (23.3%); p =0.01, odds ratio (OR) 1.5, 95% confidence interval (CI) 1.0-2.3. Seropositivity for both phases IgG, compatible with recent or persistent infection, was detected in 55 [20.1%] vs. 22 [9.7%]; p <0.001, OR 2.3, 95% CI 1.3-3.9. High phase II IgG antibodies compatible with active or recent infection (titres 1:160) were detected in 27 [9.6%] vs. 7 [3.1%]; p< 0.002, OR 3.4, 95% CI 1.4-8.0, respectively. Q fever was diagnosed in 14 (5.1%) cases. The risk of abortion associated with serological markers of active or recent Q fever in pregnant women was measurable and noticeable in this population, and accounted for 12% (95% CI, 4-21%). 

 

Background: Insights into long-term mortality and especially causes of death after initial recovery from an episode of community-acquired pneumonia (CAP) may help in determining optimal preventive measures in such patients.Methods: Prospective observational cohort studies were conducted to compare cause-specific long-term mortality rates for 356 patients recovered from CAP with the general Dutch population (16.3 million) between 2003 and 2007. The Dutch Municipal Public Records Database and death certificates were used to determine cause-specific mortality rates up to 7 years after discharge.Results: In patients recovered from CAP, cumulative 1-, 5-, and 7-year mortality rates were 17%, 43%, and 53%, respectively, compared to 4%, 19%, 24% for an age and sex matched population reference cohort. Overall, patients recovered from CAP had significantly higher long-term mortality than matched population controls (rate ratio [RR] 3.6; P <0.001).In the years after an episode of CAP, malignancy (27%), chronic obstructive pulmonary disease (COPD) (19%) and cardiovascular disease (16%) were the most frequent causes of death. Only 6% died of pneumonia as compared to 3.2% in the general population.Conclusions: After initial recovery from an episode of CAP, long-term mortality rates are more than three times higher than in the general population. Causes for long-term mortality were mostly comorbidity related and significantly different as compared to the general population. After an episode of CAP, optimizing treatment of comorbidities as treatment for COPD might have impact on improved long-time survival rates. 

 

Studies suggest that infection with highly prevalent Pseudomonas aeruginosa clones in cystic fibrosis is associated with unfavourable clinical outcome. We studied clinical characteristics of patients infected with a recently described highly prevalent P. aeruginosa clone (ST406) in two CF centres in the Netherlands. Multi Locus Sequence Typing data were available for 219 patients, of whom 40 (18.3%) were infected with ST406 and 179 with other sequence types. ST406 infection was independently associated with age, having a sibling with ST406 and use of inhaled antibiotics, but not with unfavourable clinical outcome, suggesting that high transmissibility is not necessarily associated with high virulence. 

 

The study describes the epidemiological investigations undertaken in one of Krakow’s city hospitals, which led to the detection of the source of infection and of the routes of transmission of a group A streptococcus (Streptococcus pyogenes), using FISH as a rapid method for detecting S.pyogenes carriage in the medical personnel involved. Four patients from the gynaecology department and 2 patients from the surgery department presented with clinical signs of infection. Detailed characteristics of the S.pyogenes strain isolated from the hospitalized patients and from one person from the medical personnel including the emm gene as well as the genes coding for superantigens, are described in this study. All patients (4 confirmed and 2 probable cases) survived; the operating theatre aid turned out to be an S. pyogenes carrier and the source of the infections. 

 

Mannose-binding lectin (MBL) is an oligomeric serum protein that is a member of the collectin class of the C-type lectin superfamily. Its deficiency is genetically determined and infers predisposition to recurrent infections as well as increased infection severity. This correlation has been demonstrated in recurrent furunculosis caused by Staphylococcus aureus, and in pneumococcal and Candida infections. The goal of this study was to find whether there is a correlation between MBL serum levels and recurrent urinary tact infections (UTI) in pre-menopausal women. The present aged-matched double-blind controlled study was conducted on 100 pre-menopausal adult women, 50 who suffer from recurrent UTI and 50 without UTI. MBL serum levels were measured in a single serum sample for each patient using an enzyme-linked immunosorbent assay. MBL serum levels [median, max - min] were [2500 ng/mL, 4 - 12000] and [2105 ng/mL, 4 - 22800] for research and control group respectively. Results of both groups were compared and shown not to be statistically different (p = 0.4). According to these results, MBL serum levels are not associated with an increased risk for recurrent UTI in pre-menopausal women. 

 

Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods which take several days to finalize, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for more rapid and complete assessment. We report the development of a suite of Real-Time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically-relevant, aerobic pathogens, and demonstrated a high degree of sensitivity and specificity against a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared to cultures. By culture and PCR, the most common organisms were Methicillin Resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus: GBS) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the gold standard, respectively. The assays identified bacterial DNA from 10 additional organisms that were not reported by quantitative or qualitative cultures. Under optimal conditions, turnaround for PCR results is as little as 4-6 hours. Real-Time PCR is a rapid and inexpensive approach, which can be easily translated into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by Real-Time PCR of chronic wounds is warranted. 

 

The emergence of metallo-ß-lactamase (MBL)-producing Enterobacteriaceae is a serious public health concern. Producers have been repeatedly isolates from patients and long-term care facility (LTCF) residents around Bolzano, and we sought to assess their prevalence and clinical impact. All routine Enterobacteriaceae isolates from a Bolzano tertiary care hospital and associated long-term care facilities in 2008 (n=5,500) were screened for MBLs, with case details reviewed for the source patients. In total, 36 producers were obtained from 29 patients, comprising: 14 Escherichia coli, six Klebsiella pneumoniae, four K. oxytoca, four Citrobacter freundii, two Enterobacter cloacae, two Morganella morganii and single C. amalonaticus, E. aerogenes, Providencia stuartii and Proteus mirabilis. All were PCR positive for blaVIM and 25 for qnrS; 19 non-K. pneumoniae had blaSHV and one had blaCTX-M-group1; 13 were from 12 LTCF residents and 23 from 17 acute-care patients. All these patients had serious underlying diseases with prolonged hospitalization or LTCF stay; only seven had infections due to the MBL-producers, comprising four urinary infections, two catheter-related bloodstream infections and one patient with both a surgical site infection and pneumonia. Five patients had more than one MBL-producing organism. PFGE identified a cluster of six related E. coli, whilst pairs of K. pneumoniae and C. freundii isolates had >85% similarity. Transformants prepared from two isolates were shown PCR-positive for blaVIM, qnrS and blaSHV; their plasmids gave similar RFLP patterns and sequencing detected blaVIM-1, qnrS1 and blaSHV-12. 

 

The effects that immigration may have on the epidemiology of tuberculosis (TB) in a low-incidence area of Italy was investigated by determining, in autochthonous and immigrant TB patients, the molecular characters of the Mycobacterium tuberculosis complex (MTBC) isolates that may be informative on their phylogeographical origin. A total of 1080 MTBC strains, collected during a 4-year period in Tuscany from 614 Italian-born and 466 foreign-born patients, were genotyped by spoligotyping and assigned to the different phylogeographical lineages that constitute the MTBC. The autochthonous Euro-American phylogeographical lineage, that includes the spoligotype families T, Haarlem, LAM, S and X, was highly prevalent among Italian-born patients with a total of 477 cases (77.7%) and foreign-born TB patients with a total of 270 cases (57.9%); 24 Italian-born (3.9%) and 141 (30.3%) foreign-born TB cases were due to MTBC genotypic families associated to distant geographic area, i.e., EAI, Beijing, CAS, and M. africanum. Strains of M. bovis and of undefined genotype, considered all together as it is not possible to assign a specific geographic origin, accounted for 113 (18.4%) Italian cases and 55 (11.8%) foreign-born cases. A total of 79 Italian TB cases (12.9%) have been attributed to transmission from immigrants to local population. No significant contribution to drug resistance appeared to be associated to imported MTBC strains. It is concluded that at present the overall impact of imported TB on public health in the low-incidence study area is relatively modest and of the same order as other western countries. 

 

As a cause of community-acquired infections, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing E. coli isolates from pets is available in China. Detection and characterization of ESBL genes (blaCTX-M, blaSHV, and blaTEM) was conducted among 240 E. coli isolates recovered from healthy and sick pets in South China from 2007 to 2008. The clonal relatedness of ESBL-producing E. coli was assessed by PFGE. ESBL-encoding genes were identified in 97 (40.4%) out of these 240 isolates, 96 (40.0%) of them harbored CTX-M. The most common CTX-M type was CTX-M-14 (n=45) and CTX-M-55 (n=24). The recently reported CTX-M-64 was identified in 3 isolates. Isolates producing CTX-M-27, 15, 65, 24, 3 and 9 were also identified. Ten isolates carried two or three CTX-M types, with the combination of CTX-M-14 and CTX-M-55 being the most frequent (n=6). ISEcp1 was identified in the upstream region of 93 out of the 107 blaCTX-M genes (86.9%). The sequence of the spacer region (45bp) between ISEcp1 and the start codon of all blaCTX-M-55 genes (except four) were identical to that of blaCTX-M-64. No major clonal relatedness was observed among these CTX-M producers. It is suggested that the horizontal transfer of blaCTX-M genes mediated by mobile elements, contributes to the dissemination of CTX-M enzymes among E. coli isolates from pets. Our finding of high prevalence of ESBL in E.coli of companion animal origin illustrates the importance of molecular surveillance in tracking CTX-M-producing E. coli strains in pets. 

 

Corneal scrapings from 30 patients with microbial keratitis were subjected to microsporidial PCR. PCR was positive for microsporidia in 10/30 patients. The Species was identified as Vittaforma corneae by sequencing in all the ten patients. The remaining 20 patients were negative for microsporidia and showed the growth of other organisms (Acanthamoeba/Fungus/Bacteria). 

 

It is reported that bacterial colonization of the airway in neonates affect the subsequent wheezing in childhood. This study aimed to explore the impact of bacterial colonization on severity and airway inflammation of children with virus induced wheezing. Nasopharyngeal aspirates (NPA) from 68 hospitalized children with bronchiolitis and 85 children with recurrent wheezing were obtained. 11 common respiratory viruses were detected by PCR or direct fluorescence assay (DFA). Bacteria were isolated from NPA with routine culture methods. Inflammatory cell counting and concentrations of cytokines/chemokines from all of the nasopharyngeal aspirates were measured. The frequency of bacterial colonization in children with recurrent wheezing was significantly higher than that of children with bronchiolitis. Bacterial colonization with virus infection had no effect on clinical manifestations, duration of hospitalization, concentrations of cytokines/chemokines and cellularity except IL-10 in the children with bronchiolitis. However, among the cases with recurrent wheezing, children who had coexisted non-invasive bacterial colonization and virus infection presented more frequent cyanosis, longer duration of hospitalization, higher concentration of IL-10, and higher percentage of neutriphil compared with children with virus infection but without bacterial colonization.Bacterial colonization in children with virus induced wheezing were common, particularly in children with recurrent wheezing. In some extent, bacterial colonization with virus infection might be contributed to the severity of children with recurrent wheezing because of its impact on airway inflammation. 

 

Breast milk can occasionally transmit serious viral and bacterial infections to pre term infants. We present three cases of late onset neonatal sepsis, including one death, occurring in pre term infants. The likely source of the micro-organisms in all three cases was expressed breast milk. 

 

A case-control study was conducted to identify risk factors for Pneumocystis jirovecii pneumonia (PCP) in renal transplant recipients. Eleven cases of PCP were matched with 22 controls. Cases occurred a median of 18 months after transplantation, none was receiving prophylaxis. Univariate analysis showed that graft rejection, duration of steroid use, use of mTOR inhibitors, and lymphocytopenia at time of prophylaxis discontinuation were risk factors for PCP. In the multivariate model only graft rejection (0R: 8.66, p=0.017) remained significantly associated with PCP. In patients with a history of graft rejection, PCP prophylaxis should be maintained, especially among those with lymphocytopenia. 

 

Detectable low micro-neutralization antibody (anti-H5 micro-NT 1:80) to avian influenza (H5N1) is usually categorized as a false-positive result. In this prospective study of 242 intensive care unit patients admitted for severe community-acquired pneumonia the prevalence of low micro-neutralization antibody (anti-H5 micro-NT) to avian influenza was 2.4%. Prior exposure to poultry was the sole independent risk for low anti-H5 micro-NT (aOR = 42.41; 95% CI = 22.45-64.51; P <0.001). We suggest low anti-H5 micro-NT titers be interpreted in conjunction with plausible poultry, environmental, and human exposures to avian influenza (H5N1). 

 

In the aftermath of a MRSA ST22 hospital-outbreak we investigated the prevalence of long-term carriage, the efficacy of MRSA decolonization treatment (DT) and spread of MRSA to households of patients and health care workers (HCWs). Furthermore we evaluated the efficacy of repeated DT in long-term MRSA carriers.Of 250 index persons (58 HCWs and 192 patients), 102 persons (19 HCWs and 83 patients) and 67 household members accepted participation. All 169 persons were sampled from: nose, throat, wounds and devices/catheters, index persons additionally from urine. Companion animals (N=35) were sampled from nostrils and anus. Environmental sites (N=490) screened were; telephone, TV remote control, toilet flush handle, favourite chair, and skirting board beside bed.Sixteen (19%) patients, two household members but no HCWs were ST22 positive. Throat was the most frequent site of colonization. In a multivariable analysis, chronic disease (P<0.001) and throat-carriage (P<0.001) were associated with long-term MRSA carriage.MRSA was found in the environments of four long-term carriers. All animals tested were negative. MRSA positive households were decolonized using nasal mupirocin TID and daily chlorhexidine body and hair wash for five days. Throat-carriers also received fucidic acid 500 mg TID combined with rifampicin 600 mg BID or clindamycin 600 mg BID for seven days. The home environment was cleaned on day two and five. At the end of follow-up 10 of 16 long-term carriers and the two household contacts were MRSA negative.In conclusion decolonization of MRSA carriers is possible, but should include treatment of household members and environment. 

 

In order to provide a statistically based evaluation of the incidence of invasive aspergillosis (IA) over time, we applied the Cumulative Sums (CUSUM) methodology, which was developed for quality control and has already been applied for the surveillance of hospital-acquired infections. Cases of IA were recorded during a 5-year period. Incidence rates of cases assumed to be hospital acquired, i.e. nosocomial IA (NIA) were analyzed by CUSUM tests. Relationships between NIA, fungal contamination and construction or renovation works were tested by using time series methods.Between January 2002 and December 2006, 81 cases of NIA were recorded. CUSUM analysis of NIA incidence showed no significant deviation from the expected monthly number of cases until August 2005, and then the CUSUM crossed the decision limit, i.e. identified a significant increase of NIA as compared to the reference period (January 2002 - December 2004). Up to April 2006 the Learning-Curve CUSUM stayed over its limit, supporting an ongoing outbreak involving 24 patients and then it significantly decreased on May 2006. Follow-up from May 2006 indicated no out-of-control situation, supporting a return to the baseline situation. In hematology wards, significant links were found between NIA incidence and fungal contamination of several sites of each ward (mainly unprotected common sites). An environmental source of contamination could be suspected but no significant relationship was found between NIA incidence and ongoing constructions or renovations. In conclusion, CUSUM test proved well suited for real-time monitoring of NIA and early identification and follow-up of an outbreak. 

 

Classification of bloodstream infections (BSIs) as community-acquired (CA), healthcare associated (HCA), and hospital-acquired (HA) has been proposed. The epidemiology and clinical features of BSI according to that classification in tertiary (TH) and community (CH) hospitals were investigated in a prospective cohort of 821 BSI episodes from 15 hospitals (10 TH and 5 CH hospitals) in Andaluca, Spain. Eighteen percent were CA, 24% were HCA and 58% were HA. The incidence of CA and HCA BSI was higher in CH than in TH (CA: 3.9 episodes per 1,000 admissions vs 2.2, p<0.01; HCA: 5.0 vs 2.9, p<0.01), whilst incidence of HA BSI was lower (7.7 vs 8.7, p<0.01). In CA and HCA BSI, the respiratory tract was more frequently the source in CH than in TH (CA: 30% vs 15%; HCA: 20% vs 9%, p0.03). In HCA BSI, chronic renal insufficiency and tunnelled catheters were less frequent in CH than in TH (11% vs 26% and 7% vs 19%, p0.03), but chronic ulcers were more frequent (22% vs 8%, p=0.008). BSIs due to methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa were very rare in CA episodes, but extended-spectrum ß-lactamase-producing Escherichia coli (ESBLEC) caused a similar proportion of all BSIs in CA, HCA and HA episodes. Multivariate analysis revealed no significant difference in mortality rates in CH and TH. HCA infections should be considered a separate class of BSI in both TH and CH, although differences between hospitals should be considered. CA BSIs were not caused by multidrug-resistant pathogens except for ESBLEC. 

 

Fluoroquinolones are being used more frequently for the treatment of multidrug-resistant Mycobacterium tuberculosis strains. Our study was designed to determine the frequency of the emergence of ciprofloxacin-resistant strains in a university hospital, the Rabta setting in Tunisia and to characterize the molecular mutations involved for resistant phenotype. A total of 495 clinical M. tuberculosis strains obtained from January 2005 to July 2008 were investigated for ciprofloxacin by the standard proportion method, polymerase chain reaction amplification and DNA sequencing. 4 (0.8%) resistant strains were identified. Among these isolates, 2 (50%) carried a gyrA punctual mutation leading to an amino acid change other than S95T. No gyrB missense mutation was found in any of the clinical isolates.Despite, fluoroquinolone resistance is still rare in Tunisia, accurate surveillance is needed in order to prevent eventual emergence of resistance to these drugs which are essential for the successful control, and particularly in treatment of multidrug-resistant tuberculosis. 

 

Pseudomonas aeruginosa is well adapted to the hospital setting and can cause a wide array of nosocomial infections, which occasionally culminate in recalcitrant outbreaks. Here we describe the first nosocomial outbreak of infection caused by blaVIM-2 positive P. aeruginosa in Germany. In November and December 2007 highly resistant P. aeruginosa strains were isolated from the urine of eleven patients of the Department of Urology of a University Hospital. Bacterial isolates were typed with Multi-Locus-Sequence-Typing (MLST) and screened for known Metallo-Beta-Lactamase (MBL) genes with PCR. Environmental sources of transmission were tested for bacterial contamination using surveillance cultures. Furthermore, a matched case-control study was performed in search of medical procedures significantly associated with case status. Typing of recovered strains confirmed VIM-2 Metallo-Beta-Lactamase producing P. aeruginosa of Sequence Type (ST) 175 in all cases. Surveillance cultures failed to verify an environmental source of the outbreak strain. Case-control analysis revealed retrograde urography as the only exposure significantly associated with case status. Our analyses suggest the transmission of a single clone of VIM-2 MBL producing P. aeruginosa leading to the infection of 11 patients within 47 days. Events in temporal proximity to retrograde urographies seem to have facilitated infection in the majority of cases. Department-specific infection control measures including reinforced hygienic procedures during retrograde urographies quickly terminated the outbreak. 

 

Based on the increase of resistance of genital mycoplasmas to effective antibacterials with worldwide significant differences, we compared the susceptibilities of a wide range of clinical isolates over several years. The susceptibilities of 469 M. hominis (n=290) and ureaplasma (n=179) isolates, collected during 1983 and 1989-2004, to eleven antibacterials were determined by agar dilution method. Additionally, the results from the routine testing (2005-2008) were considered. Doxycycline was the most active tetracycline against ureaplasmas and M. hominis (MIC90s, 1 and 8 mg/L, respectively). Significantly more M. hominis isolates (10-13%) were resistant to tetracyclines compared to ureaplasmas (1-3%). Ofloxacin was effective against both species (>95% susceptibility). Ciprofloxacin was moderately active against M. hominis and less active against ureaplasmas (70.3 and 35.2% susceptibility). Clarithromycin and josamycin were the most potent macrolides (MIC90, 0.5 mg/L) against ureaplasmas. Erythromycin exhibited the lowest activity (MIC90, 8 mg/L) like clindamycin, non-active against ureaplasmas, but the most potent drug against M. hominis. Cross-resistance was found between the antibacterials with the higher level between tetracyclines (53-93%), macrolides and erythromycin (70-100%) and between erythromycin and ciprofloxacin (43-55%). M. hominis has become more resistant to tetracyclines and fluoroquinolones over the years 1989-2004, with no changes in 2005-2008. Ureaplasmas have become more resistant to CIP in 1997-2004, showing high resistance to ERY over the time period (1989-2008). Doxycycline is still the drug of first-choice for ureaplasmal infections and may be used by co-infection with M. hominis. Finally, this study gives recommendation for alternatives in cases of resistance towards the commonly used antimicrobials. 

 

Acute otitis media (AOM) is an inflammatory response to microbes in the middle ear, sometimes associated with rupture of the tympanic membrane. Human leukocytes produce different patterns of inflammatory mediators in vitro when stimulated with Gram-positive and Gram-negative bacteria, respectively. Here, we investigated the cytokine and prostaglandin E2 (PGE2) responses in middle ear fluids (MEFs) from children with spontaneous perforated AOM and related the levels to the presence of pathogens detected by culture (live) or PCR (live or dead). Furthermore, in vivo cytokine pattern was compared with that induced in leukocytes stimulated by dead bacteria in vitro. MEFs with culturable pathogenic bacteria contained more IL-1ß (median 110 vs <7.5 ng/ml), TNF (6.3 vs <2.5 ng/ml), IL-8 (410 vs 38 ng/ml), and IL-10 (0.48 vs <0.30 ng/ml), than culture negative fluids, irrespective of PCR findings. IL-6 and PGE2 were equally abundant (69-110 ng/ml) in effusions with live, dead or undetectable bacteria. Cytokine levels were unrelated to bacterial species and to the presence or absence of virus. Similar levels of TNF and IL-6 as found in the MEFs were obtained by in vitro stimulation of leukocytes, while 11x more IL-1ß and 3.5x more IL-8 was produced in vivo, and 22x more IL-10 was produced in vitro. A vigorous production of pro-inflammatory cytokines accompany AOM with membrane rupture regardless of causative agent, but the production seems to cease rapidly once the bacteria are killed and fragmented. IL-6 and PGE2, however, remain after bacterial disintegration and may play a role in the resolution phase. 

 

Nasopharyngeal aspirates collected during outbreak of novel influenza A (H1N1) virus in Barcelona, were tested to compare the accuracy of a rapid antigen-based rapid test (Binax) with rRT-PCR assay developed by CDC. Sensitivity, specificity and positive predictive value (PPV) of rapid test are more elevated in patients lower than 18 years old and during the acute stage of epidemic, than adult patients. 

 

Knowledge on Streptococcus agalactiae epidemiology in Portugal is limited. Therefore, we aimed to study the S. agalactiae carriage rate among Portuguese reproductive-aged women and the prevalence of antibiotic resistance and to perform the molecular characterization of the clinical isolates. S. agalactiae was recovered from 6.2% of 4269 women during 2005-2007, revealing the predominance of capsular genotypes III (35%), V (33%), Ia (16%) and II (10%). To our knowledge, this is the first report of S. agalactiae colonization rate in Portugal performed according to CDC guidelines. All isolates were susceptible to penicillin and vancomycin, whereas resistance to clindamycin and erythromycin was detected in 10% and 19% of isolates, respectively. For the latter, 53% displayed the constitutive MLSB phenotype (conferring high-level resistance to macrolides), 42% had the inducible MLSB, and M phenotype accounted for 5% of isolates. erm methylase genes were exclusively associated with MLSB phenotype isolates, whereas the M phenotype was due to the presence of mefA. MLST analysis of the genetic relatedness among isolates presenting resistance to erythromycin demonstrated a novel association between erythromycin resistance and subtype III-1/ST-19 genetic clone family.In conclusion, considering the association of genetic lineages expressing type III capsule with macrolide resistance, and its relation with invasive infection, epidemiological surveillance of this genotype is crucial. 

 

Invasive medical technology has led to an increase in the incidence of health care-associated infective endocarditis (HAIE). The aim of this study is to describe the characteristics of HAIE and to establish a comparison between health care and community-acquired episodes. HAIE was defined as either IE manifesting >48 hours after admission to hospital or IE acquired in association with a significant invasive procedure performed within 6 months before diagnosis. This is a prospective multicenter cohort study, conducted at seven hospitals belonging to the Health Service of Andalusia, in Spain. Seven hundred and ninety three cases of infective endocarditis (IE) were investigated and HAIE accounted for 16% (127 cases). Compared with community-acquired infection, patients with HAIE were older (60.1 ± 14.4 years vs. 53.6 ± 17.5) and had more co-morbidities (Charlson index number, 3.3 ± 2.3 vs. 1.8 ± 2.3) and staphylococcal infections (58.3% vs. 24.8%). Vascular manipulation was the main cause of bacteraemia responsible for HAIE (63%). Peripheral vein catheter-associated bacteraemia accounted for 32.8% of the catheter-related bacteraemia. In-hospital mortality (44.9% vs. 24.2%) was higher in the HAIE group. Septic shock (OR 2.2, 95% CI 2.9-30.2) and surgery not performed because of high surgical risk (OR 1.6, 95% CI 1.2-20) were independent predictors of mortality in the HAIE cohort. The present study demonstrates that HAIE is a growing health problem associated to high mortality. Extremely careful management of vascular accesses is needed to minimize the risk of secondary bacteremias. 

 

To analyze current data on transmission of cytomegalovirus (HCMV) via breast milk with subsequent symptomatic HCMV infection of the preterm infant and to report on long-term follow-up. A systematic literature review on transmission of HCMV via breast milk to the premature born infant was performed using Medline, Embase and Cinahl (1966 - Dec 2008) Studies were included for analysis if congenital HCMV infection was excluded and transmission via breast milk was either confirmed or highly suspicious. Twenty-six studies were included for analysis. Maternal HCMV-IgG positivity was reported to range from 51.6 to 100 percent (median 81.6%), HCMV-IgG detection in breast milk from 67 to 97.2 percent (median 80%), and HCMV-positivity of the infants from 5.7 to 58.6 percent. Symptomatic HCMV disease occurred in 0 to 34.5 percent (median 3.7%) and severe sepsis-like syndrome in 0 to 13.8 percent (median 0.7%). Data on long term outcome of preterm infants with symptomatic HCMV infection revealed a low risk for mild neurologic and cognitive sequelae without hearing impairment. Recommendations for high risk preterm infants diverged markedly.Current data report on low rates of symptomatic disease following transmission of cytomegalovirus (HCMV) via breast milk to the preterm infant without evidence of certain long-term sequelae. Results do not support a general approach by either avoidance or pasteurization of breast milk in high risk preterm infants. 

 

Capnocytophaga, a Gram-negative anaerobe that inhabits the oral cavity, has been reported to be an unusual cause of chorioamnionitis and neonatal infection.We report 5 cases of Capnocytophaga sp. infection in preterm infants (1 proven infection and 4 probable infections) and reviewed 14 previously reported cases.We suggest that Capnocytophaga sp. may be responsible for some occult causes of chorioamnionitis or preterm birth and that the prevalence of this infection may therefore be higher than previously reported. 

 

Nontuberculous mycobacteria that can not be identified to the species level by reverse line blot hybridization assays and sequencing of the 16S rDNA gene are a challenge for reference laboratories. However, the number of 16S gene sequences added to online public databases is growing rapidly and so is the number of Mycobacterium species. Therefore, we re-analyzed 178 Mycobacterium isolates with 53 previously unmatched 16S rDNA gene sequences, submitted to our national reference laboratory in the 1999 tot 2007 period. All 16S sequences were re-compared with the GenBank database and the isolates were re-identified using two commercially available identification kits, targeting separate genetic loci.Ninety-three out of 178 isolates (52%) with 20 different 16S rDNA sequences could now be assigned to validly published species. The two reverse line blot assays provided false identifications for three recently described species and we recorded 22 discrepancies in identification results between the two reverse line blot assays.Identification by reverse line blot assays underestimates the genetic heterogeneity among NTM. This heterogeneity can be clinically relevant as particular sub-groupings of species can cause specific disease types. Therefore, sequence-based identification is preferable, at least at reference laboratory level, although the exact targets needed for clinically useful results remains to be established. The number of NTM species in the environment is probably so high that unidentifiable clinical isolates should be designated a separate species status only if this is clinically meaningful. 

 

Microsporidia is an emerging ocular pathogen. In this study, we describe the seasonal trends of microsporidial keratitis. The incidence of microsporidial keratitis is increasing in India and, with a seasonal trend toward disease onset during the monsoon. 

 

Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of human and animal tuberculosis. This complex encompasses several phylogenetically-related species, namely M. tuberculosis, the main etiological agent of human tuberculosis, and M. bovis, the causative agent of bovine tuberculosis, a relevant worldwide zoonosis. Clear epidemiological evaluation towards appropriate and effective treatment requires unambiguous differentiation between MTC members. Routine diagnosis has been increasingly relying on the molecular identification of MTC members. Here we present the use of a gold-nanoparticle based approach for the sensitive, specific and fast identification of MTC and for the differentiation of M. bovis and M. tuberculosis using as target the gyrB locus. This gold-nanoprobe strategy relies on the colorimetric differentiation of specific DNA sequences induced by differential aggregation profiles in presence and/or absence of specific target hybridization. Three nanoprobes were designed and successfully used for specific identification as member of MTC, M. bovis and M. tuberculosis. 

 

We evaluated the distribution of the two known Streptococcus pneumoniae pilus encoding islets (PI-1 and PI-2) among a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). In addition, we found that: the PI positive isolates, 50% of which belong to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), correlate with the genotype (as determined by Multi Locus Sequence Typing) but not with the serotype; PI-2 was not present in the absence of Pl-1; the frequency of PI-1 was higher among antibiotic resistant isolates. 

 

The actuations of health professionals in compliance with protocols in the different phases of an influenza pandemic are essential. In order to evaluate compliance with preparedness plans, actors simulating avian influenza attended various hospital emergency departments and public health centres during the last quarter of 2007. Most centres (89%) did not respond correctly. The useful information obtained was sent to the medical and administrative staff responsible for preparedness plans. Awareness of these errors and their rectification can lead to improvements in the response to any case of influenza with pandemic potential and in the capacity to combat any other emergent or re-emergent community infection. 

 

We performed an 11-year retrospective analysis of consecutive non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates in two neighboring hospitals in the Paris area. MRSA isolates were classified according to resistance (R) to fluoroquinolones (Fq), kanamycin (K), tobramycin (T), and gentamicin (G). The yearly number of MRSA isolates (3446 in total) decreased, from approximately 350 in 1997-2002 to 212 in 2007. Four patterns (P) were found: P1 (KTGFq R, n=776), P2 (KTFq R; G susceptible [S], n=1630), P3 (Fq R; KTG S, n=397); and P4 (Fq S; any KTG, n=201). P1 predominated in 1997 (183 isolates) then dropped sharply (9 in 2007); P2 and P4 remained stable over time; and P3 increased from 13 isolates in 1997 to 72 in 2007. Patterns were significantly and positively associated with several variables, independently from year of collection: P1, age <80 years, male gender, ICU stay, and hospital onset; P3, age >80 years and stay in intermediate or long-term care wards; and P4, age <40 years, stay in an obstetric ward, and imported cases. Molecular typing of 79 isolates in 2005 and 2007 using MLST, spa type, and SCCmec showed that P1, P2, and P3 isolates were mainly clonal, whereas P4 isolates were more diverse. P1 was mainly constituted of ST247-I isolates, P2 of ST8-IVc and P3 of ST8-IVc and ST5-VI. In conclusion, the epidemiology of MRSA is changing rapidly at the local level, with phenotypically defined clones being substituted by others and with associations existing between the changes and specific populations or circumstances. 

 

Influenza vaccine provides protection against infection with matched strains, and this protection correlates with serum antibody titers. In addition to antibodies, Flu-specific CD8+ T-lymphocyte responses are important in decreasing disease severity and facilitating viral clearance. Since this response is directed at internal, relatively conserved antigens, it affords some cross-protection within a given subtype of influenza. With the possibility of a broader H1N1 Mexico outbreak in the fall of 2009, the degree of cellular immune response cross reactivity between influenza isolates is a consideration. The composition of the 2006-2007 influenza vaccine included the A/New Caledonia/20/1999 strain – a virus that has been circulating and included in vaccine preparations for 6-7 years, and two other strains (Wisconsin and Malaysia) not previously included. This combination afforded us the opportunity to determine the degree of cross reactive cellular immunity following exposure to new viral strains. We analyzed the antibody responses and the phenotype and function of the T cell response vaccine components. Our results show that antibody responses to A/New-Caledonia were already high and vaccination did not increase antibody or CTL responses. These data suggest that repeated exposures to the same influenza stain results in limited boosting of humoral and cellular immune responses. 

 

The molecular epidemiology of 33 E. coli and 81 K. pneumoniae ESBL-producing health-care associated and community-acquired isolates collected in the Helsinki district during 2000-2004 was investigated. Clonality studies, antimicrobial susceptibility and genotyping of the isolates were performed. Newly emerging CTX-M-producing E. coli and blaSHV-12-producing K. pneumoniae were detected. Clonal clusters of both species persisted throughout the study period. 

 

The optimum treatment for prosthetic joint infections has not been clearly defined. In this study, our experience in the management of acute hematogenous prosthetic joint infection (AHPJI) is presented by analyzing data from patients with AHPJI prospectively identified during a 3-year period in 9 Spanish hospitals. Fifty patients, 30 (60%) women, median age 76 years, were diagnosed with AHPJI. The median infection-free period following joint replacement was 4.9 years. Symptoms were acute in all cases. A distant previous infection and/or bacteremia was identified in 48%. Etiology: Staphylococcus aureus 19, Streptococcus spp. 14, gram-negative bacilli 12, anaerobes 2, and mixed infections 3. Thirty-four (68%) patients underwent conservative surgical approach (CSA) with implant retention and 16, prosthesis removal. At 2 years follow-up 24 (48%) were cured, 7 (14%) relapsed, 7 (14%) died, 5 (10%) had persistent infection, 5 reinfection, and 2 an unknown evolution. Overall, the failure rate was 57.8% in staphylococcal infections and 14.3% in streptococcal infections. There were no failures in patients with gram-negative bacilli infection. On multivariate analysis, CSA was the only factor independently associated with treatment failure (OR: 11.6; 95% CI, 1.29 to 104.8). We were unable to identify any factors predicting treatment failure in patients treated with CSA, although a gram-negative bacilli etiology was a protective factor. These data suggest that although conservative surgery was the only factor independently associated with treatment failure, it could be the first therapeutic management for gram-negative bacilli, streptococcal AHPJI, and for some cases with short coming S. aureus infections. 

 

We determined the mutation frequencies of the 59 nosocomial isolates of Enterobacter cloacae, and investigated their association with antimicrobial susceptibility, genotype, and history of exposure to antimicrobials. The frequencies of mutation to rifampicin resistance ranged from 5.8 × 10-9 to 8.0 × 10-6 (median, 5.0 × 10-8). Seven of the 59 (12%) isolates graded strong mutators exhibiting more than 50-fold increase in the mutation frequency relative to that of E. cloacae ATCC13047, and 30 (52%) graded weak mutators exhibiting more than 5-fold and not more than 50-fold increase in the mutation frequency. The isolates with higher grade of mutation frequency were resistant to significantly more antimicrobials (median numbers; 2, 1, and 0 agents for strong, weak, and non-mutators, respectively; P = 0.0078). The 59 isolates were classified into 36 genotypes, and all of the seven strong mutators had distinct genotypes. Mutation frequencies varied more than 102-fold within a clone. In patient-based, univariate analysis, ICU admission, dense antimicrobial exposure (glycopeptide or multiple classes) and repetitive detection of this species were significantly more common among all of the four patients from whom strong mutator was obtained. Strong mutators are highly prevalent in surgical isolates of E. cloacae. Higher mutation frequency was associated with antimicrobial resistance and repetitive detection, and may contribute to the adaptability of this species. 

 

Tigecycline (TGC) has demonstrated clinical efficacy and safety versus imipenem/cilastatin in Phase 3 clinical trials for complicated intra-abdominal infection (cIAI). The current study was a multicentre, open-label, randomised study of TGC versus ceftriaxone plus metronidazole (CTX/MET) for the treatment of patients with cIAI. Eligible subjects were randomised (1:1) to receive either an initial TGC 100 mg dose followed by 50 mg q 12 hours, or CTX 2 grams once daily plus MET 1 gram to 2 grams daily for 4 – 14 days. The primary endpoint was clinical response in the clinically evaluable (CE) population at the test of cure (TOC) assessment. Of 473 randomised subjects, 376 were CE. Among these, clinical cure rates were 70.4% (133/189) for TGC versus 74.3% (139/187) for CTX/MET (95% CI -13.1, 5.1; p=0.009 for non-inferiority). Clinical cure rates for subjects with APACHE II scores 10 were 56.8% (21/37) for TGC versus 58.3% (21/36) for CTX/MET. Microbiologic response was similar between the two treatment arms, with microbiological eradication at TOC achieved in 68.1% (94/138) of TGC-treated subjects and 71.5% (98/137) of CTX/MET-treated subjects. The most frequently reported adverse events (AEs) for both treatment arms were nausea (TGC 38.6% vs CTX/MET 27.7%) and vomiting (TGC 23.3% vs CTX/MET 17.7%). Overall discontinuation rates due to an AE were 8.9% and 4.8% in TGC and comparator subjects, respectively. The results of this study demonstrate that TGC monotherapy is non-inferior to a combination regimen of CTX/MET in treating subjects with cIAIs. 

 

Recently there has been international concern at the rapid emergence of highly pathogenic strains of Staphylococcus aureus associated with a toxin called Panton Valentine Leukocidin (PVL). In the UK these strains are believed to be rare and mainly severe. We estimate the proportion of staphylococcal infections that are caused by strains containing the PVL genes, and describe risk factors for these infections.Three hundred and ninety consecutive S. aureus clinical isolates, submitted for routine diagnostic purposes were screened for PVL genes. Risk factors for infection were identified from the patient medical record. 12.6% (95% CI 8.3 to 14.4%) of clinical isolates and 22.6% of skin and soft tissue specimens contained the genes for PVL. Methicillin-resistant S. aureus (MRSA) with PVL was rare (1.3% of all isolates) but PVL with Methicillin-sensitive S. aureus (MSSA) was common (11.3% of all specimens). PVL infection was more frequent in males (OR 2.1, 95% CI 1.0 to 4.2), and in young adults aged 20-39 years (OR 3.2, 95% CI 1.2 to 8.4). Over three-quarters of PVL positive S. aureus infections originated in patients based in the community.Community-onset PVL-associated disease is common in the UK and mainly causes skin and soft tissue infections that do not require admission to hospital. Consideration should be given to current infection control strategy, which advocates household contact screening and decolonisation on the assumption that PVL-associated disease is rare. 

 

The inability of conventional identification systems to accurately identify Gordonia species often results in misdiagnosis of infections by these rare pathogens, which require genomic sequencing for precise identification. Here we describe nine cases of the various types of infection caused by Gordonia species. From 1997 to 2008, 66 isolates (from 30 patients) of Rhodoccus isolates initially identified based on conventional biochemical methods by the Bacteriology Laboratory of National Taiwan University Hospital (NTUH) were retrospectively analyzed the accuracy of species identification. Fifteen of these isolates (from nine patients) were later confirmed as Gordonia species by using two molecular methods: PCR-restriction fragment length polymorphism (PCR-RFLP) for heat shock protein gene (hsp65) and the 16S rRNA gene sequencing analysis. G. sputi (n = 8) was the most common pathogen, followed by G. terrae (n = 7). Most of the isolates were isolated from blood (n = 11), followed by soft tissue (n = 2) and eye (n = 2). Five patients presented with bacteremia and two of them had catheter-related bloodstream infection. Two patients had soft tissue infections and another two patients had infective keratitis and conjunctivitis. The RAPD patterns for isolates from different patients had different patterns indicating that they were genetically unrelated. In conclusions, Gordonia species are difficult to identify by conventional laboratory methods but can cause human infections including bacteremia, soft tissue infection, and keratitis. Accurate identification with molecular methods is imperative for the diagnosis of infection caused by these unusual pathogens. 

 

In 46 febrile neutropenic patients who had received hematopoietic stem cell transplantation the fluorescence in situ hybridisation using peptide nucleic acid probes (PNA FISH method), Gram stain/acridine-orange leucocyte cytospin (Gram/AOLC) test, and differential time to positivity (DTP) method were performed for detection of catheter related bloodstream infections (CRBSI). Compared to DTP (detected 11 patients with CRBSI) PNA FISH as well as Gram/AOLC detected 10 out of 11 CRBSI patients resulting in a sensitivity, specificity, NPV and PPV of 91%, 100%, 97% and 100%, respectively, for PNA FISH as well as for Gram/AOLC. 

 

Binax NOW Streptococcus pneumoniae urinary antigen (ICT) is a rapid and reliable method to identify a pneumococcal aetiology of pneumonia. The aim of this study was to evaluate the attitude of clinicians in their everyday practice towards prescription of ICT and the impact of its results on the adaptation of the antibiotic therapy, when suspecting a pneumonia. From October 2007 to March 2008, we prospectively evaluated 541 consecutive inpatients who had an ICT performed in our institution. Of the 541 patients evaluated, only 233 (43%) were alleged to have a pneumonia by the treating physicians, 58 of which having a positive ICT. Among these 58 patients, 4 (7%) and 26 (45%) were treated in monotherapy by amoxicillin respectively before and after the result of the ICT (p<10-4). Although a positive ICT has led to a rise in the proportion of patients treated by amoxicillin alone, a large number remained treated by broader spectrum antibiotics. These results suggest that prescription monitoring of the ICT should be implemented along with a more efficient adherence to treatment guidelines. 

 

Staphylococcus aureus bacteraemia (SAB) is a serious infection that demands prompt clinical attention for good outcome. To assess the impact of intervention by infectious diseases physicians (IDPs) in cases with SAB, a retrospective cohort study of patients with SAB, comparing the management and outcome of patients during the initial and the later half of the intervention period, was performed in a 1240-bed, university hospital in Japan. Three hundred and forty six patients with SAB during 7-year period, from 2002 to 2008, were included, and 194 patients in the initial half of the period (from 2002 to 2005) were compared with 152 patients in the later period (from 2006 to 2008). No significant difference was seen between the 2 groups in patient’s clinical background. The proportion of methicillin resistant S. aureus was lower during the later period (56.2% vs. 43.3%; P=.02). Echocardiography was used more frequently (37.1% vs. 64.5%; P=.00). Infective endocarditis and metastatic infections were diagnosed more frequently (10.8% vs. 20.4%; P=.00). Follow-up blood cultures were obtained more regularly (52.1% vs. 73.7%; P=.00) and therapy was more frequently administered for at least 14 days (47.4% vs. 82.2%; P=.00). The 30-day mortality improved during the intervention period (25.8% vs. 16.4%; P=.04). The number of blood cultures increased annually and the number of consultations increased about 1.6 fold compared with 2002.Proactive intervention by IDPs raised awareness of optimal management of bacteraemia and improved the adherence to the standards of care, which subsequently resulted in the improvement of the outcome. 

 

Clin Microbiol InfectAbstractThe cholera burden has grown strikingly during the past 4 years, and has spread to countries previously spared by this disease. The current spread has proved especially violent, as illustrated by the recent deadly epidemics around the Lake Chad Basin, in East Africa, and in Haiti. This onset of severe cholera epidemics is part of the overall dynamic of the current seventh cholera pandemic, composed of successive epidemic waves. The current wave is attributable to new atypical El Tor strains, which spread from the Bay of Bengal to Papua in the east, Africa, and the Caribbean Sea in the west, and caused hundreds of thousands of cases and thousands of deaths during each of the last 4 years. The particular severity of the resulting epidemics is partially attributable to the specific characteristics of the atypical El Tor strain involved. Besides the abilty of El Tor to spread easily, this strain is associated with more severe clinical findings, because of elevated levels of toxin secretion resulting from a genetic content originating from classical strains. Conversely, recent studies of these deadly outbreaks raised hope by illustrating their relationship with human-borne dissemination rather than with the resurgence of environmental strains. As human-borne dissemination can be more easily targeted than ubiquitous environmental contamination, accurate and comprehensive epidemiological studies are essential to better understand the dynamics of the disease and to optimize future cholera responses. 

 

Clin Microbiol InfectAbstractWe assessed the comparative efficacy of empirical therapy with beta-lactam plus macrolide vs. beta-lactam plus doxycycline for the treatment of community-acquired pneumonia (CAP) among patients in the Australian Community-Acquired Pneumonia Study. Both regimens demonstrated similar outcomes against CAP due to either atypical’ (Chlamydophila, Legionella or Mycoplasma spp.) or typical bacterial pathogens. 

 

Clin Microbiol InfectAbstractPatients with pneumonia treated in the internal medicine department (IMD) are often at risk of healthcare-associated pneumonia (HCAP). The importance of HCAP is controversial. We invited physicians from 72 IMDs to report on all patients with pneumonia hospitalized in their department during 2 weeks (one each in January and June 2010) to compare HCAP with community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP). We analysed 1002 episodes of pneumonia: 58.9% were CAP, 30.6% were HCAP and 10.4% were HAP. A comparison between CAP, HCAP and HAP showed that HCAP patients were older (77, 83 and 80.5 years; p < 0.001), had poorer functional status (Barthel 100, 30 and 65; p < 0.001) and had more risk factors for aspiration pneumonia (18, 50 and 34%; p < 0.001). The frequency of testing to establish an aetiological diagnosis was lower among HCAP patients (87, 72 and 79; p < 0.001), as was adherence to the therapeutic recommendations of guidelines (70, 23 and 56%; p < 0.001). In-hospital mortality increased progressively between CAP, HCAP and HAP (8, 19 and 27%; p < 0.001). Streptococcus pneumoniae was the main pathogen in CAP and HCAP. Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) caused 17 and 12.3% of HCAP. In patients with a confirmed aetiological diagnosis, the independent risk factors for pneumonia due do difficult-to-treat microorganisms (Enterobacteriaceae, P. aeruginosa or MRSA) were HCAP, chronic obstructive pulmonary diseases and higher Port Severity Index. Our data confirm the importance of maintaining high awareness of HCAP among patients treated in IMDs, because of the different aetiologies, therapy requirements and prognosis of this population. 

 

Clin Microbiol InfectAbstractStaphylococcus aureus small-colony variants (SCVs) are being isolated more frequently in cystic fibrosis (CF) patients. We aimed to determine the prevalence of S. aureus SCVs and their phenotypic and genotypic properties in CF patients admitted to a university hospital. Specimens of 248 patients were examined during a period of 11 months. Colonies supposed to be SCVs were evaluated on Columbia blood agar, mannitol salt agar, and brain–heart infusion agar with 5% NaCl (BHIA 5% NaCl). Strains were confirmed by S. aureus nucA PCR. Antibiotic susceptibilities of SCVs and simultaneously isolated S. aureus strains were determined for oxacillin, gentamicin, trimethoprim–sulphamethoxazole, vancomycin, ciprofloxacin, linezolid, and tigecycline. Genetic relatedness between SCVs and normal S. aureus strains was determined with a pulsed-field gel electrophoresis (PFGE) method. S. aureus SCVs were detected in 20 of 248 patients (8.1%). The highest SCV isolation rate was obtained with MSA, followed by BHIA 5% NaCl. Auxotrophism for thymidine was demonstrated in six SCVs. The tigecycline susceptibilities of 48 SCV strains isolated in this study showed higher MIC values than those of 33 simultaneously isolated normal S. aureus strains. Whereas SCVs and normal S. aureus strains showed identical genotypes in 14 of the patients, five patients showed different genotypes. This first study from Turkey evaluating S. aureus SCVs in CF patients has indicated the importance of considering and reporting SCVs in chronic infections such as CF. The presence of SCVs will probably indicate persistent infection, and this might impact on antibiotic treatment decisions, as they are more resistant to antibiotics. 

 

Clin Microbiol InfectAbstractExtraintestinal pathogenic Escherichia coli (ExPEC) strains, a major cause of bacteraemia, typically belong to phylogenetic group B2 and express diverse accessory traits that contribute to virulence in mouse infection models. However, their high genomic diversity obscures the relationship between virulence factors and severity of infection in patients. In this study, we analysed concomitantly the strain’s expression of virulence in a mouse model, genomic determinants and the clinical severity of infection in 60 bacteraemic patients. We show that bacterial virulence based on an animal model study and virulence factor determination is not correlated with pejorative outcome of E. coli human blood infections. 

 

Clin Microbiol InfectAbstractCulture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae, Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as no known aetiology’. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower Ct values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated. 

 

Clin Microbiol InfectAbstractThis study aimed to evaluate the routine setting performance of a guideline for phenotypic detection of extended spectrum ß-lactamases (ESBLs) in Enterobacteriaceae, recommending ESBL confirmation with Etest or combination disc for isolates with a positive ESBL screen test (i.e. cefotaxime and/or ceftazidime MIC >1 mg/L or an automated system ESBL warning). Twenty laboratories submitted 443 Enterobacteriaceae with a positive ESBL screen test and their confirmation test result (74%Escherichia coli, 12%Enterobacter cloacae, 8%Klebsiella pneumoniae, 3%Proteus mirabilis, 2%Klebsiella oxytoca). Presence of ESBL genes was used as reference test. Accuracy of local phenotypic ESBL detection was 88%. The positive predictive value (PPV) of local screen tests was 70%, and differed per method (Vitek-2: 69%, Phoenix: 68%, disc diffusion: 92%), and species (95%K. pneumoniae-27%K. oxytoca). A low PPV (3%) was observed for isolates with automated system alarm but third-generation cephalosporin MICs <2 mg/L. Local ESBL confirmation had a PPV and negative predictive value (NPV) of 93% and 90%, respectively. Compared with centrally performed confirmation tests, 7% of local tests were misinterpreted. Combination disc was more specific than Etest (91% versus 61%). Confirmation tests were not reliable for P. mirabilis and K. oxytoca (PPV 33% and 38%, respectively, although NPVs were 100%). In conclusion, performance of Etests could be enhanced by education of technicians to improve their interpretation, by genotypic ESBL confirmation of P. mirabilis and K. oxytoca isolates with positive phenotypic ESBL confirmation, and by interpreting isolates with a positive ESBL alarm but an MIC <2 mg/L for cefotaxime and ceftazidime as ESBL-negative. 

 

Clin Microbiol InfectAbstractProsthetic joint infection remains one of the most devastating complications of arthroplasty. Debridement and retention of the prosthesis is an attractive management option in carefully selected patients. Despite this, there are no data investigating the cost of this management modality for prosthetic joint infections. The aim of this case–control study was to calculate the cost associated with debridement and retention for management of prosthetic joint infection compared with primary joint replacement surgery without prosthetic joint infection. From 1 January 2008 to 30 June 2010, there were 21 prosthetic joint infections matched to 42 control patients. Controls were matched to cases according to the arthroplasty site, age and sex. Cases had a greater number of unplanned readmissions (100% vs. 7.1%; p <0.001), more additional surgery (3.3 vs. 0.07; p <0.001) and longer total bed days (31.6 vs. 7.9 days; p <0.001). In addition they had more inpatient, outpatient and emergency department visits (p <0.001, respectively). For patients with prosthetic joint infection the total cost, including index operation and costs of management of the prosthetic joint infection, was 3.1 times the cost of primary arthoplasty; the mean cost for cases was Australian dollars (AUD) $69 414 (±29 869) compared with $22 085 (±8147) (p <0.001). The demand for arthroplasty continues to grow and with that, the number of prosthetic joint infections will also increase, placing significant burden on the health system. Our study adds significantly to the growing body of evidence highlighting the substantial costs associated with prosthetic joint infection. 

 

Clin Microbiol InfectAbstractAlthough the influenza A (H1N1) 2009 virus is expected to circulate as a seasonal virus for some years after the pandemic period, its behaviour cannot be predicted. We analysed a prospective cohort study of hospitalized adults with influenza A (H1N1) 2009 pneumonia at 14 teaching hospitals in Spain to compare the epidemiology, clinical features and outcomes of influenza A (H1N1) 2009 pneumonia between the pandemic period and the first post-pandemic influenza season. A total of 348 patients were included: 234 during the pandemic period and 114 during the first post-pandemic influenza season. Patients during the post-pandemic period were older and more likely to have chronic obstructive pulmonary disease, chronic kidney disease and cancer than the others. Septic shock, altered mental status and respiratory failure on arrival at hospital were significantly more common during the post-pandemic period. Time from illness onset to receipt of antiviral therapy was also longer during this period. Early antiviral therapy was less frequently administered to patients during the post-pandemic period (22.9% versus 10.9%; p 0.009). In addition, length of stay was longer, and need for mechanical ventilation and intensive-care unit admission were significantly higher during the post-pandemic period. In-hospital mortality (5.1% versus 21.2%; p <0.001) was also greater during this period. In conclusion, significant epidemiological changes and an increased severity of influenza A (H1N1) 2009 pneumonia were found in the first post-pandemic influenza season. Physicians should consider influenza A (H1N1) 2009 when selecting microbiological testing and treatment in patients with pneumonia in the upcoming influenza season. 

 

Clin Microbiol InfectAbstractClinical breakpoints are used in clinical microbiology laboratories to categorize microorganisms as clinically susceptible (S), intermediate (I) or resistant (R) dependent on the quantitative antimicrobial susceptibility as indicated by the MIC value determined in a well-defined standard test system. The laboratory report, with the designations of S, I or R for each antimicrobial agent, provides guidance to clinicians with respect to the potential use of agents in the treatment of patients, and clinical breakpoints should therefore distinguish between patients that are likely or unlikely to respond to antimicrobial treatment. In Europe, clinical breakpoints are set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), following a defined procedure. This includes evaluation of efficacy in experimental settings and clinical studies to derive pharmacodynamic targets such as the fAUC/MIC ratio or %fT > MIC required for efficacy, the pharmacokinetic properties of the agent, Monte Carlo simulations to estimate exposures of the antimicrobial agent in the target patient population and commonly used dosing regimens. The probability of target attainment is subsequently determined for a range of pharmacodynamic targets and the results from the Monte Carlo simulations. The breakpoints derived are subsequently evaluated with respect to the wild-type population of the target microorganisms, specific resistance mechanisms and other relevant data. In this paper, we provide an overview of the EUCAST process and considerations for setting pharmacokinetic/pharmacodynamic breakpoints. These are the breakpoints that in the EUCAST breakpoint tables are referred to as non-species-related breakpoints’. 

 

Clin Microbiol InfectAbstractOseltamivir or zanamivir are effective in outpatients with seasonal influenza; however, factors associated with response have been incompletely described. During the 2008/2009 epidemic, in a randomized trial for influenza A-infected outpatients, clinical (time to alleviation of flu-related symptoms) and virological (rate of patients with day 2 nasal viral load <200 cgeq/L) responses to oseltamivir or zanamivir were assessed and associated factors were determined using multivariate analysis. For oseltamivir (141 patients) and zanamivir (149 patients) median times to alleviation of symptoms were 3 and 4 days, respectively; 59% and 34% had virological response. For oseltamivir, a lower clinical response was associated with female gender (HR, 0.53; 95% CI, 0.36–0.79), baseline symptoms score >14 (HR, 0.47; 0.32–0.70), viral load 5 log cgeq/L (HR, 0.63; 0.43–0.93), and initiation of antibiotics (HR, 0.30; 0.12–0.76); a lower virological response was associated with female gender (OR, 0.45; 0.21–0.96), baseline viral load 5 log cgeq/L (OR, 0.40; 0.20–0.84) and days 0–2 incomplete compliance (OR, 0.31; 0.10–0.98). For zanamivir, virological response was associated with age 50 years (OR, 0.29; 0.10–0.85) and initiation of antibiotics at baseline (OR, 4.24; 1.07–17.50). Factors associated with lower response to neuraminidase inhibitors in outpatients appeared to be easily identifiable during routine clinical examination and, when appropriate, by nasal sampling at baseline. The unknown association between gender and response to oseltamivir was not explained by compliance. 

 

Clin Microbiol InfectAbstract16S ribosomal RNA methylase-mediated high-level resistance to 4-,6-aminoglycosides has been reported in clinical isolates of gram-negative bacilli from several countries. Three of 1534 (0.2%) isolates of Klebsiella pneumoniae and three of 734 (0.4%) Proteus mirabilis isolates from a university hospital in Athens, Greece, were positive for rmtB and highly resistant to all aminoglycosides tested (MICs 256 mg/L). Two of the K. pneumoniae rmtB-bearing isolates, were KPC-2 and OXA-10 producers and the third was a DHA-1 producer. One of the P. mirabilis isolates was a VIM-1 and OXA-10 producer and one was an OXA-10 producer. All rmtB-harbouring isolates were clonally unrelated. None of the E. coli (n = 1398) and Enterobacter spp. (n = 414) isolates were positive for armA, rmtA, rmtB, rmtC or rmtD. 

 

Clin Microbiol InfectAbstractThis study aimed to develop a modular, diagnostic algorithm for extended spectrum ß-lactamase (ESBL) detection in Enterobacteriaceae. Clinical Enterobacteriaceae strains (n = 2518) were screened for ESBL production using Clinical and Laboratory Standards Institute (CLSI) breakpoints for third-generation cephalosporins and by synergy image detection (clavulanic acid/extended-spectrum cephalosporins). Isolates screening positive for ESBL (n = 242, 108 by critical CLSI diameters alone, five by double disk synergy test (DDST) alone, and 129 by both critical diameters and DDST) and 138 ESBL screening negative isolates (control group) were investigated by molecular methods considered to be the reference standard (multiplex CTX-M type PCR, TEM and SHV type sequence characterization). One hundred and twenty-four out of 242 Enterobacteriaceae isolates screening positive for ESBL were confirmed to be ESBL positive by the reference standard, the majority of them in E. coli, K. pneumoniae and E. cloacae (94, 17 and nine isolates, respectively). Prevalence of ESBL production ranged from <1% for P. mirabilis to 4.7%, 5.1% and 6.6%, for K. pneumoniae, E. cloacae and E. coli, respectively. Combining CLSI ceftriaxone and cefpodoxime critical ESBL diameters was found to be the most sensitive phenotypic screening method (sensitivity 99.2%). Combining critical diameters of cefpodoxime and ceftriaxone with DDST for cefpodoxime resulted in a sensitivity of 100%. For phenotypic confirmation, combining the CLSI recommended combined disk test (CDT) for ceftazidime and cefotaxime amended with a cefepime CDT was highly sensitive (100%) and specific (97.5%). With respect to the studied population, the diagnostic ESBL algorithm developed would have resulted in sensitivity and specificity of 100%. The corresponding flow chart is simple, easy to use, inexpensive and applicable in the routine diagnostic laboratory. 

 

Clin Microbiol InfectAbstractThe aim of this study was to determine if severity assessment tools (general severity of illness and community-acquired pneumonia specific scores) can be used to guide decisions for patients admitted to the intensive care unit (ICU) due to pandemic influenza A pneumonia. A prospective, observational, multicentre study included 265 patients with a mean age of 42 (±16.1) years and an ICU mortality of 31.7%. On admission to the ICU, the mean pneumonia severity index (PSI) score was 103.2 ± 43.2 points, the CURB-65 score was 1.7 ± 1.1 points and the PIRO-CAP score was 3.2 ± 1.5 points. None of the scores had a good predictive ability: area under the ROC for PSI, 0.72 (95% CI, 0.65–0.78); CURB-65, 0.67 (95% CI, 0.59–0.74); and PIRO-CAP, 0.64 (95% CI, 0.56–0.71). The PSI score (OR, 1.022 (1.009–1.034), p 0.001) was independently associated with ICU mortality; however, none of the three scores, when used at ICU admission, were able to reliably detect a low-risk group of patients. Low risk for mortality was identified in 27.5% of patients using PIRO-CAP, but above 40% when using PSI (I–III) or CURB65 (<2). Observed mortality was 13.7%, 13.5% and 19.4%, respectively. Pneumonia-specific scores undervalued severity and should not be used as instruments to guide decisions in the ICU. 

 

Clin Microbiol InfectAbstractComparisons of bloodstream infection (BSI) rates between neonatal intensive-care units (NICUs) should take into account differences in babies’ vulnerability and invasive procedures that can introduce infection. Our aim was to investigate which risk factors recorded in routine records should be adjusted for when NICUs are compared. This was a retrospective cohort study using routine records for two London NICUs. We analysed rates of BSI with Poisson regression models. The level of neonatal care used by the National Health Service was the strongest predictor of BSI incidence. The rate ratios for BSI, adjusted for birthweight, inborn/outborn status, and postnatal age, were 3.15 (95% CI 2.01–4.94) for intensive care and 6.58 (95% CI 4.18–10.36) for high-dependency care, relative to special care. Total parenteral nutrition was significantly associated with BSI incidence, but explained less of the variance among babies than level of care. A case–control study with the same dataset gave similar results. Further multicentre studies are required to confirm our predictive model. Until then, we recommend that comparisons of BSI rates between NICUs should include adjustments for level of care, birthweight, inborn/outborn status, and postnatal age, with the use of routinely recorded standardized measures in hospital administrative data. 

 

Clin Microbiol InfectAbstractThe aim of the study was to describe the characteristics of acute hepatitis E in Greater Cairo. Patients with acute hepatitis E were identified through a surveillance of acute hepatitis using the following definition: recent (<3 weeks) onset of fever or jaundice, alanine aminotransferase at least three times the upper limit of normal (uln), negative markers for other causes of viral hepatitis and detectable hepatitis E virus (HEV) RNA. Comparison of the liver tests between acute hepatitis E and hepatitis A virus (HAV), case–control analysis (four sex-matched and age-matched (±1 year) HAV controls per case) to explore risk factors and phylogenetic analyses were performed. Of the 17 acute HEV patients identified between 2002 and 2007, 14 were male. Median age was 16 years (interquartile range 13–22). Compared with HAV (n = 68 sex-matched and ±1 year age-matched), HEV patients had higher bilirubin (mean (SD) 10.9 (5.7) uln versus 7.5 (4.4) uln, p 0.05) and aspartate aminotransferase levels (38.6 (27.1) uln versus 18.3 (18.1) uln, p 0.02). Co-infection (hepatitis C virus RNA or hepatitis B surface (HBs) -antigen positive/IgM anti-hepatitis B core (HBc) anitgen negative) was diagnosed in four patients. In univariate matched analysis (17 cases, 68 matched controls), HEV cases were more likely to live in a rural area than HAV controls (matched OR 7.9; 95% CI 2.0–30.4). Of the 16 isolates confirmed as genotype 1, 15 belonged to the same cluster with 94–98.5% identity in the open-reading frame 2 region. Our findings documented the sporadic nature of HEV in Greater Cairo, characterized a large number of Egyptian HEV genotype 1 strains and identified living in a rural area as a potential risk factor for infection. 

 

Clin Microbiol InfectAbstractThe swine-origin H1N1 influenza A virus (pH1N1(2009)) started to circulate worldwide in 2009, and cases were notified in a number of sub-Saharan African countries. However, no epidemiological data allowing estimation of the epidemic burden were available in this region, preventing comprehensive comparisons with other parts of the world. The CoPanFlu-Mali programme studied a cohort of 202 individuals living in the rural commune of Dioro (southern central Mali). Pre-pandemic and post-pandemic paired sera (sampled in 2006 and April 2010, respectively) were tested by the haemagglutination inhibition (HI) method. Different estimates of pH1N1(2009) infection during the 2009 first epidemic wave were used (increased prevalence of HI titre of 1/40 or 1/80, seroconversions) and provided convergent attack rate values (12.4–14.9%), the highest values being observed in the 0–19-year age group (16.0–18.4%). In all age groups, pre-pandemic HI titres of 1/40 were associated with complete absence of seroconversion; and geometric mean titres were <15 in individuals who seroconverted and >20 in others. Important variations in seroconversion rate existed among the different villages investigated. Despite limitations resulting from the size and composition of the sample analysed, this study provides strong evidence that the impact of the pH1N1(2009) first wave was more important than previously believed, and that the determinants of the epidemic spread in sub-Saharan populations were quite different from those observed in developed countries. 

 

Clin Microbiol InfectAbstractThe emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays. 

 

Clin Microbiol InfectAbstractWe analysed the 12-week virological response to protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) therapy in 1108 patients carrying B or non-B human immunodeficiency virus (HIV)-1 subtypes with matched resistance mutation patterns. Response rates were not significantly different for non-B and B subtypes stratified for treatment status (51.5% vs. 41.5% in nave patients; 46.7% vs. 38.7% in experienced patients) or regimens (46.9% vs. 39.7% with PI; 56.7% vs. 40% with NNRTI). No difference in response was detected in patients harbouring B and non-B subtypes with any resistance profile. Further studies are advisable to fully test this approach on larger datasets. 

 

Clin Microbiol InfectAbstractIn this paper we report on the development and validation of two different methods for posaconazole quantification from serum samples, HPLC/UV and bioassay. Both methods have been validated according to international guidelines and were also applied to the analysis of 61 trough serum samples from treated patients. A good correlation between both methods was observed. The HPLC method, more laborious and expensive, was demonstrated to be more accurate, precise and faster (analytical range 0.125–16 g/mL, accuracy between -2.48 and 3.70% and precision between 2.77 and 5.93%, with an analytical run time of 11 min), making it a valuable tool for reference laboratories that centralize high numbers of samples. The microbiological method, however, is simple and offers sufficient precision and accuracy (analytical range 0.125–16 g/mL, accuracy between -8.10 and 3.77% and a precision between 4.52 and 10.07%), to be used to monitor posaconazole. It may be a valid alternative to chromatographic methods in clinical laboratories without specialized facilities. 

 

Clin Microbiol InfectAbstractWe report data concerning the detection of fungal DNA directly from lysis–centrifugation blood culture to assess its value in the detection of fungaemia in 86 of the 347 patients admitted to the neonatal intensive-care unit between January 2009 and December 2010. The sensitivity and specificity of the PCR were 87.5% and 98.5%, respectively, with a positive predictive value of 93.3% and a negative predictive value of 97.1%. Detection of fungal DNA directly from blood culture Isolator 1.5 microbial tubes, without prior cultivation, is a promising approach for the rapid detection of Candida spp. in neonates with suspected candidaemia. 

 

Clin Microbiol InfectAbstractOf 109 clinical Escherichia coli isolates from two major tertiary hospitals in Lagos (University Teaching Hospital and the National Orthopaedic Hospital Igbobi), 14 (12.8%) extended-spectrum beta-lactamse (ESBL) producers were characterized using PCR and sequencing, ERIC-PCR and multilocus sequence typing. All ESBL-producing isolates encoded only the CTX-M-15 gene. Clonal group ST131 (35.7%) was the predominant ST, followed by ST617 (28.6%). Isolated cases of other sequence types were also observed. Plasmid-mediated quinolone resistance genes qnrA, qnrB1 and aac-(6)-lb-cr were detected among these ESBL isolates of different clonal groups. This is the first description of the clonality of CTX-M-15-producing E. coli from Nigeria. The presence of diverse clonal lineages shows the continuing potential for genetic diversification and emergence of new epidemic strains. 

 

Clin Microbiol InfectAbstractWe investigated the presence of qnrC and qnrD among 756 non-replicate Enterobacteriaceae isolated in Italy, selected for being non-susceptible to fluoroquinolones and/or resistant to third-generation cephalosporins. Four Proteus mirabilis and one Morganella morganii (0.66% of the total) presented a qnrD gene, located in a 2687-base-pair plasmid that was entirely sequenced. The plasmid is un-typable, and contains no known coding region other than qnrD. That the qnrD gene was found in four unrelated P. mirabilis and in one M. morganii isolate might suggest a frequent association of this gene with the tribe Proteeae. 

 

Clin Microbiol InfectAbstractImprovement in hand hygiene (HH) compliance has been associated with a decrease in the incidence of hospital-acquired infection (HAI) and hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) infection/colonization. We aimed to evaluate the impact of a multimodal intervention in medical wards on HH compliance, alcohol-based hand rub (AHR) consumption and incidence of HAI and HA-MRSA. A before–after intervention study and an assessment 1 year later were conducted in three internal medicine wards. HH compliance during routine patient care was monitored using the WHO HH observation method. AHR consumption was registered. HAI incidence was actively sought during the PRE and POST periods. HAI risk factors were prospectively recorded and incidence density was calculated. A total of 825 patients were prospectively followed in the PRE period and 868 patients in the POST period. We observed 1531 opportunities for HH in PRE and POST periods and 450 1 year later. HH compliance improved from 54.3% to 75.8% (p 0.005) and remained 75.8% at follow-up. AHR consumption increased from 10.5 to 27.2 L/1000 hospital-days and 31.5 L/1000 hospital-days at follow-up. Incidence density of HAI was 6.93 and 6.96/1000 hospital-days in the PRE and POST intervention periods, respectively. HA-MRSA incidence density was 0.92 in the PRE period vs. 0.25/1000 hospital-days in the POST period (p 0.2) and 0.15/1000 hospital-days (p 0.1) 1 year later. A sustained increase in AHR consumption was followed by an improvement in HH compliance after a multimodal campaign. A trend for lower incidence density of new hospital-acquired MRSA was detected in the POST intervention and follow-up periods. 

 

Clin Microbiol InfectAbstractOnly limited data are available on the development of neutralizing antibodies (NAB) in patients with chronic hepatitis C (CHC) treated with pegylated interferon- (PEG-IFN-). The aim of this study was to evaluate the immunogenicity of PEG-IFN- when administered to CHC patients who had or had not previously received standard IFN- therapy. In addition, the specificities of NAB, together with the ability of leucocyte (LE) -IFN- to re-establish therapeutic responsiveness in NAB-positive patients, were evaluated. NAB were assessed using a quantitative, standardized, virus-induced cytopathic effect assay. The seroconversion rate to PEG-IFN- was higher in patients who had received previous standard IFN- treatment than in those treated exclusively with PEG-IFN-. Also, NAB produced during PEG-IFN- therapy were unable to neutralize LE-IFN- entirely, even though they can neutralize several IFN- subtypes. In addition, the results indicate that a change to LE-IFN- therapy can be associated with restoration of clinical responses in NAB-positive patients who had become resistant after showing an initial response to PEG-IFN- treatment. This study emphasizes the importance of evaluating NAB development in CHC patients who become resistant to PEG-IFN- treatment, and suggests management alternatives for patients who develop NAB. 

 

Clin Microbiol InfectAbstractAlthough tick-borne encephalitis (TBE) has been recognized in Europe for more than 70 years and has been the topic of numerous reports, information on the involvement of facial nerves in the course of the disease is limited. Our study conducted at a single medical centre revealed that facial nerve involvement in the course of TBE in Central Europe is (i) infrequent—it was found in only 11 of 1218 (0.9%) consecutive adult patients diagnosed with TBE; (ii) manifests with unilateral or rarely bilateral peripheral facial palsy (PFP) (nine and two patients, respectively); (iii) appears late in the course of acute illness—in our patients 10–20 days after the onset of the meningoencephalitic phase of TBE, and often after defervescence (in 8/11 patients; 6–13 days after normalization of body temperature); (iv) develops more often in patients with more severe illness, i.e. more frequently in those with encephalitic than in those with meningitic clinical presentation, and more commonly in patients with monophasic than biphasic illness; and (v) has a favourable outcome—our patients had a clinically complete recovery from PFP within 7–90 (median 30) days after its onset. Moreover, the finding of Borrelia infection in 3/11 (27.3%) patients (diagnosis of confirmed Lyme neuroborreliosis was established in 1/11 patients and two patients fulfilled criteria for possible Lyme neuroborreliosis) suggests that in countries where TBE and Lyme borreliosis are endemic, concomitant infection with Borrelia burgdorferi sensu lato should be considered and searched for in patients who develop PFP in the course of TBE. 

 

Clin Microbiol InfectAbstractScrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation. 

 

Clin Microbiol InfectAbstractClofazimine has shown activity against Mycobacterium tuberculosis, including multidrug-resistant strains in vitro and in animal studies. However, clinical experience with clofazimine in multidrug-resistant tuberculosis (MDR-TB) is scarce. We reported our clinical experience with 39 MDR-TB patients treated with combination regimens that included clofazimine. From January 2008 to March 2011, 39 patients received clofazimine for the treatment of MDR-TB in Shanghai Pulmonary Hospital. Patients had isolates resistant to a median of six drugs (range, 2–11 drugs). Of the 39 cases, 36 had cavitary changes noted on initial chest radiograph or chest computed tomography, and positive sputum-smear microscopy results at the time of MDR-TB diagnosis. At data censure, 15 of the 39 patients had successful therapy, with at least five consistently negative cultures documented for the final 12 months of treatment. Eleven continued to receive treatment. There were no deaths. Thirteen patients had a poor outcome, including four defaults and nine treatment failures. Culture conversion occurred in 22 cases at a median of 12 weeks. Side-effects occurred in 34 patients, mainly including skin discolouration, ichthyosis and gastrointestinal adverse events. No patients reported significant toxicity likely to be attributable to clofazimine therapy. Adverse events were managed by combinations of dose adjustment and symptom management. In our experience, clofazimine was well tolerated and may have efficacy in the treatment of MDR-TB. 

 

Clin Microbiol InfectAbstractHepatic Candida infection (HCI; known as chronic disseminated candidosis or CDC) is a distinct form of disseminated Candida infection with predominant involvement of the liver. Diagnosis of HCI is usually made on clinical suspicion together with multiple lesions in liver on ultrasound (US), CT and/or MRI scan. Fungal elements may not always be visible in liver tissue and mycological culture is frequently negative, making the evidence for proven fungal disease difficult. We studied a novel commercially available low-cost and density-array (LCD) chip technique for a molecular diagnosis of HCI. This is a two-step procedure with PCR amplification after DNA extraction followed by hybridization on a small chip provided by the manufacturer (Fungi 2.1, Chipron GmbH). The analysis of DNA from 45 fungal control strains showed an excellent specificity and sensitivity. The DNA from 11 liver biopsies of patients with haematological malignancies suffering from CDC was analysed on the LCD chip and overall 11 fungal pathogens could be detected in eight liver biopsies, supporting the clinical diagnosis of HCI/CDC. Analysis of liver biopsies from controls was negative for fungal DNA in all samples studied. In conclusion, the novel LCD chip technique examined in our study was able to detect fungal pathogens in liver biopsies from patients with haematological malignancies and suspected HCI/CDC but was negative in control biopsies. 

 

Clin Microbiol InfectAbstractCarbapenem resistance is increasingly being reported among Acinetobacter species, and results mostly from the expression of acquired carbapenem-hydrolysing oxacillinases (CHDLs). Several Acinetobacter species intrinsically possess chromosomal CHDL genes: Acinetobacter baumannii (blaOXA-51), Acinetobacter radioresistens (blaOXA-23), and Acinetobacter lwoffii (blaOXA-134). We aimed to identify the progenitors of novel CHDL-encoding genes for identification of potential reservoirs. We performed PCR screening using degenerated internal primers designed from a sequence alignment of the known CHDLs (OXA-23, OXA-40, OXA-51, OXA-58, OXA-134, and OXA-143) applied to a collection of 50 Acinetobacter strains belonging to 23 different species. Two strains of Acinetobacter johnsonii, one strain of Acinetobacter calcoaceticus and two strains of Acinetobacter haemolyticus were found to harbour, respectively, the totally novel blaOXA-211-like, blaOXA-213-like and blaOXA-214-like genes. In addition, the complete genomes of those three species available in GenBank, i.e. one A. johnsonii genome, four A. calcoaceticus genomes, and one A. haemolyticus genome, were analysed and found to be positive for the presence of blaOXA211-like, blaOXA-213-like and blaOXA-214-like genes, respectively. The ß-lactamases OXA-211, OXA-213 and OXA-214 are diverse, with amino acid identities ranging from 53% to 76%, as compared with the naturally occurring OXA-51-like CHDL from A. baumannii. These ß-lactamases showed a peculiar hydrolysis profile, including mostly penicillins and carbapenems. Regarding blaOXA-23 in A. radioresistens and blaOXA-134 in A. lwoffii, these genes were not expressed (or expressed at a non-significant level) in their host. Detection of these ß-lactamase genes might be used as a useful tool for accurate identification of these Acinetobacter species. 

 

Clin Microbiol InfectAbstractEUCAST expert rules have been developed to assist clinical microbiologists and describe actions to be taken in response to specific antimicrobial susceptibility test results. They include recommendations on reporting, such as inferring susceptibility to other agents from results with one, suppression of results that may be inappropriate, and editing of results from susceptible to intermediate or resistant or from intermediate to resistant on the basis of an inferred resistance mechanism. They are based on current clinical and/or microbiological evidence. EUCAST expert rules also include intrinsic resistance phenotypes and exceptional resistance phenotypes, which have not yet been reported or are very rare. The applicability of EUCAST expert rules depends on the MIC breakpoints used to define the rules. Setting appropriate clinical breakpoints, based on treating patients and not on the detection of resistance mechanisms, may lead to modification of some expert rules in the future. 

 

Clin Microbiol InfectAbstractThe effects of antibiotic timing on outcomes of patients with community-acquired pneumonia (CAP) are controversial. Moreover, no information is available regarding this issue in healthcare-associated pneumonia (HCAP). We aimed to determine the impact of antibiotic timing on 30-day mortality of patients with CAP and HCAP. Non-immunocompromised adults admitted to hospital through the emergency department (ED) with community-onset pneumonia were prospectively observed from 2001 to 2009. Patients who received prior antibiotics were excluded. Of 1593 patients with pneumonia who were analyzed, 1274 had CAP and 319 HCAP. The mean time from patient arrival at the ED until antibiotic administration was 5.8 h (standard deviation (SD) 3.5) in CAP and 6.1 h (SD 3.8) in HCAP (p 0.30). Mortality was higher in patients with HCAP (5.5% vs. 13.5%; p <0.001). After adjusting for confounding factors in a logistic regression analysis, the antibiotic administration 4 h was not associated with decreased 30-day mortality in patients with CAP (odds ratio (OR) 1.12, 95% confidence interval (CI) 0.57–2.21) and in patients with HCAP (OR 0.59, 95% CI 0.19–1.83). Similarly, antibiotic administration 8 h was not associated with decreased 30-day mortality in CAP (OR 1.58, 95% CI 0.64–3.88) and HCAP patients (OR 0.59, 95% CI 0.19–1.83). In conclusion, antibiotic administration within 4 or 8 h of arrival at the ED did not improve 30-day survival in hospitalized adults for CAP or HCAP. 

 

Clin Microbiol InfectAbstractTo better recognize the pathogenicity of ocular Demodex mites, we analysed Bacillus oleronius infection in patients with Demodex-related chronic blepharitis. The studies were conducted on 68 adult patients, in whom ophthalmological and parasitological tests permitted the distinction of a group of 38 patients with a diagnosis of Demodex-related chronic blepharitis (group 1, including a subgroup 1a with moderate blepharitis and a subgroup 1b with severe blepharitis) and a group of 30 healthy individuals (group 2). In every person studied six eyelashes were epilated from each eye and the number of Demodex per eyelash was scored. In parallel, bacterial culture and isolation allowed their phenotypic and molecular identification. The drug sensitivity of the isolates was tested using E-tests. Intensity of Demodex infestation showed no significant differences between subgroups 1a and 1b. From the epilated eyelashes 23 bacterial isolates were obtained, identified as being B. oleronius. All the studied strains were sensitive to ciprofloxacin, doxycycline and gentamicin. The Demodex mite represents an independent aetiopathogenetic factor in blepharitis. In parallel, the parasite may act as a carrier of B. oleronius bacteria, which most probably function as a co-pathogen in the development of severe forms of blepharitis. 

 

Clin Microbiol InfectAbstractStreptococcus pneumoniae pilus islet-1 (PI-1)-encoded pilus enhances in vitro adhesion to the respiratory epithelium and may contribute to pneumococcal nasopharyngeal colonization and transmission. The pilus subunits are regarded as potential protein vaccine candidates. In this study, we sought to determine PI-1 prevalence in carried pneumococcal isolates and explore its relationship with transmissibility or carriage duration. We studied 896 pneumococcal isolates collected during a longitudinal carriage study that included monthly nasopharyngeal swabbing of 234 infants and their mothers between the ages of 1 and 24 months. These were cultured according to the WHO pneumococcal carriage detection protocol. PI-1 PCR and genotyping by multilocus sequence typing were performed on isolates chosen according to specific carriage and transmission definitions. Overall, 35.2% of the isolates were PI-1-positive, but PI-1 presence was restricted to ten of the 34 serotypes studied and was most frequently associated with serotypes 19F and 23F; 47.5% of transmitted and 43.3% of non-transmitted isolates were PI-1-positive (OR 1.2; 95% CI 0.8–1.7; p 0.4). The duration of first-ever infant pneumococcal carriage was significantly longer with PI-1-positive organisms, but this difference was not significant at the individual serotype level. In conclusion, PI-1 is commonly found in pneumococcal carriage isolates, but does not appear to be associated with pneumococcal transmissibility or carriage duration. 

 

Clin Microbiol InfectAbstractVaricella zoster virus (VZV) is a leading cause of acute viral encephalitis but little is known about its clinical, biological and imaging features. Furthermore, the most favourable treatment regimen has not been determined. We studied a prospective cohort of 20 HIV-negative patients presenting with acute VZV encephalitis caused by primary infection or reactivation. VZV was identified in 16 of 20 cases by PCR detection of the DNA in the cerebrospinal fluid. The four remaining cases occurred during or soon after a VZV rash. The median age of the 17 adults was 76 (19–86) years; the three other patients were children (0.5–5 years). Three patients were immunocompromised. Nine adult patients presented with a rash. Eighteen patients presented with fever and an acute encephalitic syndrome: diffuse brain dysfunction, focal neurological signs, seizures and cranial nerve palsies. Three patients presented with either ventricular or subdural haemorrhage, one with myelitis, and one with asymptomatic stenosis of the middle cerebral artery. The imaging was either normal or revealed non-specific abnormalities such as cortical atrophy but no evidence of stroke. All patients were given acyclovir at various dosages and durations but the case fatality rate remained high (15%) and sequelae were frequently observed either at discharge or at follow-up 3 years later. 

 

Clin Microbiol InfectAbstractIn recent years, we have successfully established a novel method of haploidentical haematopoietic stem cell transplantation (HSCT) without in vitro T-cell depletion. This study was aimed at analysing the incidence and risk factors of invasive fungal infection (IFI) with this transplantation method. The study comprised 291 patients who had undergone haploidentical HSCT from 1 January 2007 to 31 December 2008. IFI was diagnosed according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group 2002 criteria, and only proven or probable cases of IFI were regarded as true cases. A total of 39 patients were documented as having IFI, including four proven cases and 35 probable cases. The median time of diagnosis was 26 days (range: 6–405 days) after transplantation. The cumulative incidence rates of IFI at 40 days, 1 year, 2 years and 3 years after transplantation were 8.25%, 13.1%, 13.4% and 13.4%, respectively. Multivariate analysis identified platelet engraftment time (>17 days) (p 0.027; hazard ratio (HR) 2.432; 95% CI 1.105–5.355), a high risk of underlying disease (p 0.001; HR 2.916; 95% CI 1.515–5.611) and grade III–IV acute graft-versus-host disease (p 0.019; HR 2.407; 95% CI 1.154–5.022) as risk factors for IFI. The incidence rates of IFI in patients with no, one, two or three risk factors at 3 years after transplantation were 4.48%, 7.86%, 29.6% and 23.1%, respectively. In conclusion, IFI is an important complication following haploidentical HSCT without in vitro T-cell depletion. 

 

Clin Microbiol InfectAbstractThe closely related members of the Acinetobacter baumannii (Ab) group (A. baumannii, A. pittii and A. nosocomialis) are difficult to identify with phenotypic tests in diagnostic laboratories. Genotypic identification methods require special skills and most do not provide rapid results. The aim of this study was to investigate the ability of MALDI-TOF MS to identify members of the Ab group. Sixty epidemiologically unrelated Acinetobacter spp. isolates were investigated by MALDI-TOF MS: 18 A. baumannii, 17 A. pittii, 18 A. nosocomialis and seven additional isolates representing other Acinetobacter spp. All strains were verified by ARDRA, rRNA intergenic spacer (ITS), recA sequencing and blaOXA-51. MALDI-TOF MS correctly identified all the genomic strains but erroneously identified A. nosocomialis as A. baumannii because there was no reference strain within the Bruker database. Peak analysis of individual spectra from representative strains of each member of A. baumannii, A. pittii and A. nosocomialis suggested enough differences between their protein signatures to allow accurate identification using MALDI-TOF MS. Inclusion of specific signature profiles for A. nosocomialis within the Bruker database allowed the correct identification of this genomic species. MALDI-TOF MS spectra can be used as a fast, simple and reliable method to identify members of the Ab group. The rapid and accurate identification of clinically significant Acinetobacter strains will improve insight into their epidemiology and allow for targeted therapeutic and infection control measures against clinically important strains. 

 

Clin Microbiol InfectAbstractA low level of CD4+ lymphocyte cells makes end-stage HIV/AIDS patients highly susceptible to microbial infections. We have adopted the next generation sequencing method to identify the spectrum of bacterial plasma and viral elements that might be present in these patients. The HIV/AIDS plasma microbiome was dominated by bacterial elements in the taxonomical order Pseudomonadales, while healthy people carried fewer bacterial DNA in the plasma. We have found that many of the bacterial elements in HIV/AIDS plasma are similar to those of the microbes found in the human gut, suggesting potential acquisition of microbial elements from the gut. The HIV/AIDS and normal plasma DNA virome shared some similarities in the presence of common ubiquitous eukaryotic viruses. The normal DNA virome was mainly composed of viruses from Anelloviridae. In contrast, the HIV/AIDS DNA virome contained a large proportion of bacteriophages, endogenous retroviruses and a non-human virus. In addition, several sequences, which might belong to novel bacteria or endogenous retroviruses, were identified. Taken together, the use of high-throughput sequencing technology in unveiling microbial metagenomics may facilitate future research in combating HIV/AIDS and its associated microbial complications. 

 

Clin Microbiol InfectAbstractHistorically, Haemophilus influenzae (Hi) serotype b (Hib) caused most invasive Haemophilus infections worldwide, mainly in children. In 1989 routine childhood vaccination against Hib was initiated in Iceland. We conducted a population-based study of all patients in the country with Haemophilus spp. isolated from sterile sites (n = 202), from 1983 to 2008. Epidemiology, clinical characteristics of the infections and serotypes of the isolates were compared during the pre-vaccination (1983–1989) and post-vaccination era (1990–2008). Following the vaccination, the overall incidence of Hib decreased from 6.4 to 0.3/100 000 per year (p <0.05) whereas the incidence did not change significantly for infections caused by Haemophilus sensu lato not serotype b, hereafter referred to as non-type b Hi (0.9 vs 1.2, respectively). The most frequent diagnosis prior to 1990 was meningitis caused by Hib, which was subsequently replaced by pneumonia and bacteraemia caused by non-type b Hi. Most commonly, non-type b Hi were non-typeable (NTHi; 40/59), followed by Hi serotype f (14/59) and Hi serotype a (3/59). Pregnancy was associated with a markedly increased susceptibility to invasive Haemophilus infections (RR 25.7; 95% CI 8.0–95.9, p <0.0001) compared with non-pregnant women. The case fatality rate for Hib was 2.4% but 14% for non-type b Hi, highest at the extremes of age. Hib vaccination gives young children excellent protection and decreases incidence in the elderly due to herd effect in the community. Replacement with other species or serotypes has not been noted. Pregnant women are an overlooked risk group. 

 

Clin Microbiol InfectAbstractIn Denmark, several laboratories use PCR as a routine diagnostic method for Legionnaires’ disease, and almost all PCR-positive samples are investigated by culture. From 1993 to 2010, isolates of Legionella species other than Legionella pneumophila were obtained from respiratory samples from 33 patients, and from 1997 to 2010, 42 isolates of Legionella non-pneumophila species were obtained and saved from water samples from 39 different sites in Denmark. Macrophage infectivity potentiator gene (mip) sequencing was used as a reference method to identify the Legionella non-pneumophila species. Only one of the 75 isolates did not meet the acceptance criterion of a similarity of 98% to sequences in the database. The species distribution between clinical and environmental isolates varied. For the former, four species were detected, with Legionella bozemanae and Legionella micdadei predominating (both 44%). For the latter, eight species were detected, with Legionella anisa predominating (52%). The distribution among the Danish clinical isolates was different from the general distribution both in Europe and outside Europe, where L. bozemanae and Legionella longbeachae are the most commonly found clinical Legionella non-pneumophila species. The 75 isolates were also investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): 64 were correctly identified, with a score of 2.0; eight had a score of <2.0, but only two of these were wrongly identified; and three gave no results with MALDI-TOF MS. Both mip sequencing and MALDI-TOF MS are robust methods for Legionella species identification. 

 

Clin Microbiol InfectAbstractThe purpose of the present study was to evaluate the significance of shortening the antibiotic treatment duration in prosthetic joint infections (PJI) treated with debridement, antibiotics and implant retention (DAIR). In April 2006 we shortened the total antibiotic treatment duration in total knee arthroplasty (TKA) PJIs from 6 months to 3 months and in total hip arthroplasty (THA) PJIs from 3 months to 2 months. All patients with TKA or THA PJI treated with DAIR between February 2001 and August 2009 were reviewed retrospectively. There were 132 patients treated with DAIR, of whom 86 (65%) completed the antibiotic therapy and were therefore eligible for comparison concerning the length of antibiotic treatment. There were 32 (37%) THA and 54 (63%) TKA PJIs in the comparison. The treatment succeeded in 34 (89.5%) patients treated with longer antibiotic treatment and in 42 (87.5%) of those treated with shorter antibiotic treatment (p 0.78). Our conclusion is that if the patient completes the antibiotic therapy, treatment duration of 3 months in TKA PJIs and 2 months in THA PJIs is as good as longer antibiotic treatment of 6 months or 3 months, respectively, in patients treated with DAIR. 

 

Clin Microbiol InfectAbstractThe aim of this study was to evaluate the clinical characteristics, predictors and outcomes of pneumococcal pneumonia developing pulmonary complications and the distribution of pneumococcal serotypes. It was a prospective study including all adult patients admitted to the Hospital Clinic of Barcelona, Spain (2001–2009) with the diagnosis of pneumococcal pneumonia. Microbiological investigation was systematically performed, including antimicrobial susceptibility and serotype distribution (only invasive strains isolated during 2006–2009). Complicated pneumonia was defined as the presence of one or more pulmonary complications: pleural effusion, empyema, or multilobar infiltrates. We included 626 patients, and 235 (38%) had the following pulmonary complications: pleural effusion, 122 (52%); empyema, 18 (8%); and multilobar infiltration, 151 (64%). Forty-six (20%) patients had more than one complication. Patients with pulmonary complications showed a higher rate of intensive-care unit admission (34% vs. 13%, p <0.001), a higher rate of shock (16% vs. 7%, p <0.001), a longer length of stay (9 days vs. 6 days, p <0.001), and a lower rate of penicillin resistance (14% vs. 25%, p 0.013), but similar mortality (9% vs. 8%). No significant differences were observed in the serotype distribution between complicated and uncomplicated pneumonia. Chronic obstructive pulmonary disease (COPD) (OR 0.38, 95% CI 0.23–0.63; p <0.001) was a protective factor against pulmonary complications, whereas chronic liver disease (OR 3.60, 95% CI 1.71–7.60; p 0.001), admission C-reactive protein level 18 mg/dL (OR 2.77, 95% CI 1.91–4.00; p <0.001) and admission creatinine level >1.5 mg/dL (OR 2.01, 95% CI 1.31–3.08; p 0.001) were risk factors for pulmonary complications. Complicated pneumonia was characterized by a more severe clinical presentation, but was not associated with increased mortality. Resistance to antibiotics was lower in complicated cases. No significant differences were observed in the serotype distribution between complicated and uncomplicated pneumonia. In the multivariate analysis, COPD was a protective factor against pulmonary complications. 

 

Clin Microbiol InfectAbstractAll organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely. 

 

Clin Microbiol InfectAbstractWe sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae. 

 

Clin Microbiol Infect 2011AbstractPatients with severe chronic obstructive pulmonary disease (COPD) are at higher risk of developing invasive pulmonary aspergillosis (IPA). However, there are limited data for this disease. To evaluate risk factors and the clinical characteristics of IPA in COPD patients, we conducted a hospital-based, retrospective case-control study of 30 COPD patients with IPA and 60 COPD control patients without IPA. Patients in the case group were significantly more likely to have concurrent co-morbidities than controls. Of the IPA patients, 65.4% had worsening radiological findings vs. 11.4% in the control group (p <0.001). IPA in COPD was associated with a higher proportion of mechanical ventilation (43.3% vs. 5%; p <0.001), a longer hospital stay duration (45.8 ± 39.1 days vs. 18.4 ± 11.8 days; p <0.001), and higher mortality (43.3% vs. 11.4%; p <0.001). Systemic use of steroids in the stable phase, treatment with three or more antibiotics during hospitalization and antibiotic treatment longer than 10 days were independent risk factors associated with IPA. COPD patients with obvious dyspnoea, antibiotic-resistant lower respiratory tract infection and repeated detection of Aspergillus in sputum should be considered for the possibility of IPA. 

 

Clin Microbiol InfectAbstractNo sufficiently powered trial has examined two antimicrobials in acute osteoarticular infections of childhood. We conducted a prospective, multicentre, quasi-randomized trial in Finland, comparing clindamycin with first-generation cephalosporins. The age of patients ranged between 3 months and 15 years, and all cases were culture-positive. We assigned antibiotic treatment intravenously for the first 2–4 days, and continued oral treatment with clindamycin 40 mg/kg/24 h or first-generation cephalosporin 150 mg/kg/24 h in four doses. Surgery was kept to a minimum. Subsiding symptoms and signs and normalization of C-reactive protein (CRP) level were preconditions for the discontinuation of antimicrobials. The main outcome was full recovery without further antimicrobials because of an osteoarticular indication during 12 months after therapy. The intention-to-treat analysis comprised 252 children, 169 of whom were analysed per-protocol: 82 cases of osteomyelitis, 80 of septic arthritis, and seven of osteomyelitis–arthritis. Staphylococcus aureus strains (all methicillin-sensitive) caused 84% of the cases. Except for one non-serious sequela during convalescence in both groups, and two late infections caused by dissimilar agents in one child, all patients recovered. The entire courses (medians) of clindamycin and cephalosporin lasted for 23 and 24 days, respectively. CRP normalized in both groups in 9 days. The patients were discharged, on average, on day 10. Loose stools were reported less often (1%) in the clindamycin group than in the cephalosporin group (7%), but two clindamycin recipients developed rash. Clindamycin or a first-generation cephalosporin, administered mostly orally, perform equally well in childhood osteoarticular infections, provided that high doses and administration four times daily are used. As most methicillin-resistant staphylococci remain clindamycin-sensitive, clindamycin remains an option instead of costly alternatives. 

 

Clin Microbiol Infect 2011AbstractThe European Organization for Research and Treatment of Cancer and the Mycosis Study Group (EORTC-MSG) radiological definitions of invasive pulmonary aspergillosis (IPA) may lack diagnostic sensitivity. We evaluated applying less restrictive radiological criteria, when supported by specific microbiological findings, to define IPA in acute myeloid leukaemia (AML), lymphoproliferative diseases (LD) and allogeneic stem cell transplant (allo-SCT) patients. Overall, 109 consecutive episodes of proven/probable IPA in 56 AML, 31 LD and 22 allo-SCT patients diagnosed from February 2006 through to January 2011 were considered. IPA was diagnosed with EORTC-MSG criteria (control group, 76 patients) or without prespecified radiological criteria (study group, 33 patients). The latter differed from the former by the inclusion of patients with pulmonary infiltrates not fulfilling the three EORTC-MSG IPA specific findings of dense, well-circumscribed lesions with or without halo sign, air crescent sign or cavity. All the analysed clinical and mycological characteristics, 3-month response to antifungal therapy and 1- and 3-month cumulative survival were comparable in the control and study groups in AML, LD and allo-SCT patients. Seventeen of 33 (51.5%) patients of the study group fulfilled EORTC-MSG radiological criteria at subsequent imaging performed a median of 15 days (range, 6–40 days) after documentation of the pulmonary infection. Our study seems to confirm the possibility of revising the EORTC-MSG criteria by extending the radiological suspicion of IPA to less specific chest computerized tomography scan findings when supported by microbiological evidence of Aspergillus infection in high-risk haematological patients. 

 

Clin Microbiol InfectAbstractWhether patients whose catheter tip grows Staphylococcus aureus but who have no concomitant bacteraemia should receive antimicrobials remains an unresolved issue. However, a proportion of patients with catheter tips colonized by S. aureus have no blood cultures taken because of low suspicion of sepsis and the meaning of this microbiological finding is unknown. We have analysed all catheter tips growing S. aureus during a 6-year period and have selected patients without blood cultures taken 7 days before or after central vascular catheter removal. Patient’s evolution was classified into good and poor outcome. Poor outcome was defined as S. aureus infection within 3 months after catheter withdrawal or death in the same period with no obvious cause. Patients with good and poor outcomes were compared to assess whether antimicrobial therapy influenced evolution. Sixty-seven patients fulfilled our inclusion criteria and five (7.4%) had a poor outcome. The administration of early anti-staphylococcal therapy had no impact on the outcome of this population (p 0.99). The only factor independently associated with a poor outcome was the presence of clinical signs of sepsis when the catheter was removed (OR 20.8; 95% CI 2.0–206.1; p 0.009). Our data suggest that patients with central vascular catheter tips colonized with S. aureus should be closely monitored for signs and symptoms of ongoing infection, but if these are not present then antimicrobial therapy does not seem justified. 

 

Clin Microbiol InfectAbstractRepeated isolation of multidrug-resistant Acinetobacter baumannii (MDRAB) from respiratory secretions poses a great challenge for infection control. We conducted a retrospective case–control study to evaluate the efficacy and adverse effect of inhaled colistin methanesulfonate (CMS) in the eradication of MDRAB from the respiratory tract. Patients who were admitted to Taipei Veterans General Hospital between February 2009 and June 2010, had at least two sets of monomicrobial culture of MDRAB from respiratory secretions, and remained in hospital for at least 14 days after the first isolation of MDRAB (index day) were included. Patients who received intravenous CMS were excluded. Patients who received CMS inhalation for 3 days were selected as cases whereas the controls were matched for age and Acute Physiology and Chronic Health Evaluation II score. Thirty-nine cases and controls were identified. The duration of CMS inhalation was 10.9 ± 3.6 days. The use of inhaled CMS was the only independent factor associated with the eradication of MDRAB within 14 days after the index day (OR 266.33; 95% CI 11.26–6302.18, p <0.001), and shortened the duration of MDRAB recovery from the respiratory tract by 13.3 ± 1.45 days. The adverse effects were similar for both groups. The increase of colistin minimal inhibitory concentrations in the last isolate compared with the index isolate from the same patient did not differ between the two groups. In conclusion, our study demonstrated that inhaled CMS enhanced the eradication of MDRAB from the respiratory tract without significant clinical adverse effect or impact on colistin resistance. 

 

Clin Microbiol InfectAbstractStaphylococcus aureus bacteraemia (SAB) is a leading cause of mortality and morbidity in both nosocomial and community settings. The objective of the study is to explore epidemiological characteristics and predisposing risk factors associated with healthcare-associated (HCA) and community-acquired (CA) SAB, and to evaluate any differences in mortality and efficacy of initial antimicrobial therapy on treatment outcome. We conducted a two-part analysis. First, a triple case–control study in which groups of HCA SAB with onset 48 h after hospital admission (HCA 48 h), HCA SAB with onset <48 h of hospital admission (HCA <48 h), and CA SAB were compared with controls. Second, a cohort study including all patients with SAB was performed to identify factors associated with in-hospital mortality. SAB was diagnosed in 165 patients over the study period (January 2007 to December 2007). Five variables were independently associated with HCA 48 h SAB: presence of central venous catheter, solid tumour, chronic renal failure, previous hospitalization and previous antibiotic therapy. Significant risk factors for HCA <48 h SAB were: Charlson Comorbidity Index 3, previous hospitalization, living in long-term care facilities and corticosteroid therapy. Factors independently associated with CA SAB were: diabetes mellitus, HIV infection and chronic live disease. Patients with HCA <48 h SAB were significantly more likely to receive initial inadequate antimicrobial treatment than patients with CA or HCA 48 h SAB (44.8% versus 33.3% and 31.5%, respectively). Logistic-regression analysis identified three variables as independent predictors of mortality: presentation with septic shock, infection with methicillin-resistant S. aureus, and initial inadequate antimicrobial treatment. More than half of patients with SAB have MRSA strains and presentation with septic shock, and inappropriate empirical therapy was associated with increased mortality. 

 

Clin Microbiol InfectAbstractThe importance of diarrhoeagenic Escherichia coli (DEC) in Africa is poorly understood, and is unknown in Burkina Faso. This study investigated the occurrence of five major DEC pathogroups in primary cultures of stool samples from 658 Burkinabe children under 5 years old using 16-plex PCR for virulence-associated genes. At least one DEC pathogroup was detected in 45% of 471 children with diarrhoea and in 29% of 187 children without diarrhoea (p <0.001). More than one DEC pathogroup was detected in 11% of children with and 1% of children without diarrhoea (p <0.001). Enteroaggregative E. coli was the most common pathogroup in both children with diarrhoea (26%) and children without diarrhoea (21%). Enteropathogenic E. coli and enterotoxigenic E. coli were detected significantly more often in children with diarrhoea (16% and 13%) than in children without diarrhoea (5% and 4%; p <0.001 for both pathogroups). Shiga toxin-producing E. coli and enteroinvasive E. coli were detected only in children with diarrhoea (2% and 1%, respectively). Diarrhoeagenic E. coli, especially enteropathogenic and enterotoxigenic, may be important, unrecognized causes of childhood diarrhoea in Burkina Faso. 

 

Clin Microbiol InfectAbstractLeishmaniasis is endemic in Europe and the prevalence of latent infection in the Mediterranean region is high. Reports describing opportunistic leishmaniasis in European patients treated with tumor necrosis factor (TNF) alpha antagonist drugs are rapidly accumulating. For other granulomatous infections, risk of opportunistic disease varies by mode of TNF-alpha antagonism. This study explores whether this may also be the case for leishmaniasis. We ascertained the relative frequency of exposure to different TNF antagonist drugs among published cases of opportunistic leishmaniasis in Europe and compared this with the prescription of these drugs in Europe. We found that risk of opportunistic leishmaniasis is higher in patients receiving anti-TNF monoclonal antibodies (infliximab or adalimumab) compared with patients treated with the TNF-receptor construct etanercept. Clinicians may want to consider these observations, which suggest that etanercept should be favoured over anti-TNF monoclonal antibodies in individuals living in or visiting areas endemic for leishmaniasis until evidence from prospective research is available. A European adverse event reporting system is required to identify rare opportunistic infections associated with immunosuppressive and immunomodulatory biotherapies. 

 

Clin Microbiol InfectAbstractThe impact of recent changes in and discrepancies between the breakpoints for cephalosporins and other antimicrobials, as determined by CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST), was analysed in patients with bloodstream infections caused by extended-spectrum ß-lactamase (ESBL) producing Escherichia coli in Spain, was analysed. We studied a cohort of 191 episodes of bloodstream infection caused by ESBL-producing E. coli in 13 Spanish hospitals; the susceptibility of isolates to different antimicrobials was investigated by microdilution and interpreted according to recommendations established in 2009 and 2010 by CLSI, and in 2011 by EUCAST. Overall, 58.6% and 14.7% of isolates were susceptible to ceftazidime, and 35.1% and 14.7% to cefepime using the CLSI-2010 and EUCAST-2009/2011 recommendations, respectively (all isolates would have been considered resistant using the previous guidelines). Discrepancies between the CLSI-2010 and the EUCAST-2011 recommendations were statistically significant for other antimicrobials only in the case of amikacin (98.4% versus 75.9% of susceptible isolates; p <0.01). The results varied depending on the ESBL produced. No significant differences were found in the percentage of patients classified as receiving appropriate therapy, following the different recommendations. Four out of 11 patients treated with active cephalosporins according to CLSI-2010 guidelines died (all had severe sepsis or shock); these cases would have been considered resistant according to EUCAST-2011. In conclusion, by using current breakpoints, extended-spectrum cephalosporins would be regarded as active agents for treating a significant proportion of patients with bloodstream infections caused by ESBL-producing E. coli. 

 

Clin Microbiol InfectAbstractInfants under 3 months of age with fever without source (FWS) generally undergo a full, invasive septic evaluation to exclude invasive bacterial infection (IBI). Enterovirus (EV) infections are mostly banal and self-limiting and show a high prevalence rate at this age. We aimed to investigate the prevalence of IBI in EV-infected and uninfected infants under 3 months of age with FWS. This was a prospective observational cohort study of infants aged <90 days who were admitted because of FWS. As per protocol, blood and urine analysis and culture were obtained in all cases, and RNA EV from blood and/or cerebrospinal fluid samples was determined by real-time PCR. Three hundred and eighty-one previously healthy infants with FWS were included. EV infection was diagnosed in 64 children (16.8%; 95% confidence interval, 13.2–20.9%) and showed an uneventful evolution in all cases. Laboratory markers of infection were consistently lower in EV-infected patients; only one case of IBI (1.6%) was observed in an EV-infected patient as compared with 25.2% in EV-negative infants (p <0.001). Intravenous antibiotic use and length of stay were no different in EV-infected and uninfected patients. In our study, febrile infants (<90 days) diagnosed with EV infection showed a low risk of IBI when compared with uninfected patients. The systematic investigation of EV infection in young infants with FWS may allow a more conservative approach to the management of these patients. Further studies on this diagnostic approach are needed. 

 

Clin Microbiol InfectAbstractIn the 1950s an unusually virulent and transmissible penicillin-resistant Staphylococcus aureus clone harbouring Panton–Valentine leukocidin (PVL) genes, known as phage type 80/81 and subsequently identified as multilocus sequence type (ST) 30, emerged and caused serious infections in hospitals and the community. We describe an outbreak of skin infections caused by a PVL-positive, methicillin-susceptible S. aureus (MSSA) strain of ST1472, related to phage type 80/81, in three associated occupational centres. After identification of the first patient an active case-finding strategy was initiated among the three centres. Epidemiological and clinical features were indistinguishable from outbreaks currently caused by community-acquired methicillin-resistant S. aureus. The S. aureus was cultured and identified from nasal swabs and skin lesions by conventional methods; PVL was detected using a PCR assay. Pulsed-field gel electrophoresis and DNA-array-based genotyping were applied to MSSA isolates. MSSA was identified in nasal swabs from 49 of 133 individuals (37%). A single pulsed-field gel electrophoresis pattern, belonging to ST1472 (CC30) and PVL positivity, were detected in 20 individuals, including eight of 18 skin cultures, i.e. 15% of the screened individuals were colonized by the epidemic strain. Nasal and cutaneous decontamination with 5% nasal mupirocin ointment and 2% aqueous chlorhexidine was implemented for all individuals. Patients with active skin infections were treated with a first-generation cephalosporin. General recommendations were made to prevent cross-transmission. No new cases were reported over the following 90 days. 

 

Clin Microbiol InfectAbstractWe evaluated whether quantitative PCR (qPCR) and (13)-ß-d-glucan assays could be used to differentiate Pneumocystis pneumonia (PCP) from Pneumocystis jirovecii colonization in immunocompromised patients with pulmonary infiltrates. A total of 40 bronchoalveolar lavage samples and 107 induced sputum samples from 147 patients who were suspected of having PCP were obtained for PCR detection of P. jirovecii. Diagnoses of definite PCP, probable PCP, pneumonia with P. jirovecii colonization (colonization) and pneumonia without colonization (non-colonization) were made in 11, 42, 15 and 60 patients, respectively. A PCP diagnosis was undetermined in 19 patients. The copy numbers, determined using qPCR, were significantly higher in definite PCP and probable PCP patients than in colonized patients. The area under the receiver-operating characteristic curve (AUC), sensitivity and specificity for discriminating definite PCP from colonization were 0.96, 100.0% and 80.0%, respectively, at a cut-off value of 1300 copies/mL. The values for discriminating probable PCP from colonization were 0.71, 66.7% and 73.3%, respectively, at a cut-off value of 340 copies/mL. ß-d-glucan levels were significantly higher in patients with both definite PCP and probable PCP than in colonized patients. The AUC, sensitivity and specificity for discriminating definite PCP were 0.91, 100.0% and 80.0%, respectively, at a cut-off value of 15.6 pg/mL. The values for discriminating probable PCP were 0.78, 76.2% and 73.3%, respectively, at a cut-off value of 6.0 pg/mL. Both qPCR and the ß-d-glucan assay displayed high accuracy for discriminating colonization from definite PCP and displayed moderate accuracy for discriminating colonization from probable PCP. 

 

Clin Microbiol InfectAbstractThe Netherlands is known for its low methicillin-resistant Staphylococcus aureus (MRSA) prevalence. Yet MRSA with no link to established Dutch risk factors for acquisition, MRSA of unknown origin (MUO), has now emerged and hampers early detection and control by active screening upon hospital admittance. We assessed the magnitude of the problem and determined the differences between MUO and MRSA of known origin (MKO) for CC398 and non-CC398. National MRSA Surveillance data (2008–2009) were analysed for epidemiological determinants and genotypic characteristics (Panton–Valentine leukocidin, spa). A quarter (24%) of the 5545 MRSA isolates registered were MUO, i.e. not from defined risk groups. There are two genotypic MUO groups: CC398 MUO (352; 26%) and non-CC398 MUO (998; 74%). CC398 MUO needs further investigation because it could suggest spread, not by direct contact with livestock (pigs, veal calves), but through the community. Non-CC398 MUO is less likely to be from a nursing home than non-CC398 MKO (relative risk 0.55; 95% CI 0.42–0.72) and Panton–Valentine leukocidin positivity was more frequent in non-CC398 MUO than MKO (relative risk 1.19; 95% CI 1.11–1.29). Exact transmission routes and risk factors for non-CC398 as CC398 MUO remain undefined. 

 

Clin Microbiol InfectAbstractNeisseria gonorrhoeae resistance to cephalosporins, the currently recommended treatment, and treatment failures with cefixime have been reported worldwide. The purposes of the present study were (i) to examine the susceptibility of N. gonorrhoeae isolates isolated in Italy from 2006 through 2010 to cefixime (n = 293) taking into account both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical And Laboratory Standards Institute (CLSI) criteria for categorization; (ii) to determine the contribution to decreased/resistant susceptibility of mutations in the penA, mtrR, ponA and porB1b genes in a subsample of isolates; and (iii) to genotype the isolates showing decreased susceptibility or resistance to cefixime, by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and by pulsed-field gel electrophoresis (PFGE) to identify the predominant genotypes. Minimum inhibitory concentrations (MICs) were determined by the E-test and agar dilution method on 293 isolates and results were interpreted according to both EUCAST 2010 (MIC R >0.12 mg/L) and CLSI 2008 (MIC R >0.25 mg/L) criteria. All isolates showed full susceptibility to ceftriaxone, whereas those with a MIC for cefixime 0.125 mg/L were on the increase from 2008 through 2010. The same penA gene alterations were found among isolates with MICs close to the EUCAST breakpoint as the resistant ones, and they belong to ST1407. Seven isolates, belonging to various sequence types, showed a different por allele, though similar to the por 908 allele present in ST1407. PFGE divided strains ST1407 into two main groups confirming their genetic relationship. 

 

Clin Microbiol InfectAbstractWe assessed the incidence of hearing loss and its relationship with clinical characteristics and pneumococcal serotypes in adults surviving pneumococcal meningitis. We analysed hearing loss in 531 adults surviving pneumococcal meningitis included in two prospective nationwide cohort studies performed from April 1998 through to October 2002 and March 2006 through to January 2009. Hearing loss was evaluated on admission and discharge for all patients. Severe hearing loss was assessed by pure tone average on audiology and corrected for age, or by the combination of hearing loss on discharge and a score on the Glasgow Outcome Scale below 5, which could not be explained by other neurological sequelae. A total of 531 episodes of pneumococcal meningitis with non-lethal outcome were included. Predisposing conditions for pneumococcal meningitis were present in the majority of patients (64%), most commonly otitis (36%). Hearing loss was present at discharge in 116 patients (22%) and was classified as mild in 53% and severe in 47%. Hearing loss was related to otitis (odds ratio [OR], 2.58; 95% confidence interval [CI], 1.66–4.02; p < 0.001) and inversely related to serotype 23F infection (OR, 0.36; 95% CI, 0.13–0.98; p = 0.025), but not with parameters of disease severity or indicators of cerebrospinal fluid inflammation severity. Meningitis due to pneumococcal serotype 3 was associated with the highest rate of hearing loss. Hearing loss frequently complicates pneumococcal meningitis. Risk factors for hearing loss were infection with pneumococcal serotype 23F and otitis, but not disease severity. Otitis and resulting perilympathic inflammation contribute to meningitis-associated hearing loss. 

 

Clin Microbiol InfectAbstractAlthough the estimate of the incidence of sepsis following transrectal ultrasound-guided prostate biopsy (TRUSPB) is low, fluoroquinolone-resistant infections after prostate biopsy are being increasingly noted. This study was aimed at determining the prevalence of faecal carriage of fluoroquinolone-resistant Escherichia coli strains before TRUSPB and at evaluating potential predisposing risk factors. The incidence of sepsis after prostate biopsy was determined, and our routine practice for antibiotic prophylaxis for TRUSPB was evaluated. A prospective study was conducted in 342 consecutive patients undergoing prostate biopsy between December 2009 and July 2010. Before TRUSPB, a rectal swab was cultured. The correlation between the presence of fluoroquinolone-resistant strains and plausible risk factors was investigated by the use of a questionnaire. Of the 236 patients included, 22.0% (52/236) harboured ciprofloxacin-resistant E. coli strains. The use of fluoroquinolones in the 6 months before biopsy was associated with an increased risk of faecal carriage of fluoroquinolone-resistant E. coli strains (p <0.01). Faecal carriage of fluoroquinolone-resistant E. coli strains was an important risk factor for infectious complications after TRUSPB (p <0.01). In conclusion, a significant number of patients have faecal carriage of fluoroquinolone-resistant E. coli strains (22.0%) before TRUSPB. The use of fluoroquinolones in the previous 6 months before biopsy is a risk factor for faecal carriage of fluoroquinolone-resistant E. coli strains and for infectious complications after TRUSPB. Hence, the universal administration of fluoroquinolones should be reconsidered. 

 

Clin Microbiol InfectAbstractThe increase in the number of methicillin-resistant Staphylococcus aureus (MRSA) infections in children has prompted paediatricians to broaden th empirical treatment of common community-onset (CO) infections in children in several countries. Most European countries have reported low rates of CO-MRSA infection, but limited data on paediatric CO-MRSA infections are available. A prospective study was conducted from January 2002 to December 2004 in Brussels. CO-MRSA was defined as MRSA first detected by culture within 48 h of admission or in outpatients. Clinical and epidemiological data were recorded. CO-MRSA strains were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Staphylococcal chromosomal cassette mec, toxin (Panton–Valentin leukocidin (PVL), toxic shock syndrome toxin 1, and Eta/b), enterotoxin and antibiotic resistance genes were detected by PCR. The antibiotic resistance phenotype was determined by disk diffusion. S. aureus was isolated in 1681 children. Among these, 107 harboured MRSA. Fifty-one children were colonized or infected by CO-MRSA, 20% of whom had no healthcare exposure. Twelve infants <3 months old and five cystic fibrosis patients were colonized. None of the 22 infected patients (59% with acute otitis media and 36% with skin and soft tissue infections (SSTIs)) required hospitalization. Two-thirds of them failed to respond to empirical antibiotic therapy. The 37 characterized CO-MRSA strains were genetically diverse. Most of them had healthcare-associated genotypes. Only six strains were PVL-positive, all of which were ciprofloxacin-susceptible and more common in children with SSTIs (p 0.001). CO-MRSA remains uncommon in our paediatric population. So far, there is no need to modify the empirical treatment of common S. aureus infections. Monitoring of MRSA rates in S. aureus CO infections remains mandatory, and further investigation is warranted to establish the source of colonization in young infants. 

 

Clin Microbiol InfectAbstractAtypical Toxoplasma gondii strains, unrelated to archetypal clonal lineages (I, II, III), have been reported more frequently over the last decade in areas other than Europe and North America. A newly described form of toxoplasmosis, Amazonian toxoplasmosis’ (AT), has been reported since 2002 in French Guiana. It is characterized by severe cases and atypical strains linked to a neotropical forest-based cycle. We report on the cases of AT that required intensive care management. We performed a prospective observational study on hospitalized adults in the Intensive Care Unit (ICU) from 2002 to 2008. Clinical and laboratory data, microbiological findings and outcomes were recorded. Data, including the ICU simplified acute physiology score and the pneumonia severity index, were calculated. Epidemiological risk factors for AT were assessed through questionnaires. Eleven non-immunodeficient patients were admitted to the ICU in Cayenne for life-threatening pneumonia associated with disseminated toxoplasmosis. Mechanical ventilation was necessary in seven patients, four of whom required immediate orotracheal intubation. Cardiac and ophthalmological abnormalities were found in five and four patients, respectively. One patient died from multiple organ failure. The genetic characterization of Toxoplasma DNA using six microsatellite markers revealed unique and atypical genotypes in eight patients. All patients presented epidemiological risk factors for AT. In French Guiana, significant T. gondii-related infectious syndrome associated with the lungs, a high level of LDH activity and the reported risk factors for AT was strongly suggestive of disseminated toxoplasmosis with a possible trend toward life-threatening pneumonia. 

 

Clin Microbiol InfectAbstractA multicentre, case–control study was conducted to assess risk factors and patient outcomes of bacteraemia caused by Enterobacteriaceae producing extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs). One hundred and five and 20 patients with bacteraemia caused by ESBL-producing and KPC-producing organisms were matched to controls who had bacteraemia caused by non-ESBL/KPC-producing organisms, respectively. Independent risk factors for ESBL production included admission from a nursing home (OR 4.64; 95% CI 2.64–8.16), chronic renal failure (OR 2.09; 95% CI 1.11–3.92), the presence of a gastrostomy tube (OR 3.36; 95% CI 1.38–8.18), length of hospital stay before infection (OR 1.02; 95% CI 1.01–1.03), transplant receipt (OR 2.48; 95% CI 1.24–4.95), and receipt of antibiotics with Gram-negative activity in the preceding 30 days (OR 1.76; 95% CI 1.00–3.08). Twenty-eight-day crude mortality rates for patients infected with ESBL-producing or KPC-producing organisms and controls were 29.1% (34/117) and 19.5% (53/272), respectively (OR 1.70; 95% CI 1.04–2.80). On multivariate analysis, inadequate empirical therapy (OR 2.26; 95% CI 1.18–4.34), onset of bacteraemia while in the intensive-care unit (OR 2.74; 95% CI 1.47–5.11), Apache II score (OR 1.17; 95% CI 1.12–1.23) and malignancy (OR 2.66; 95% CI 1.31–5.41) were independent risk factors for mortality. CTX-M was the most common ESBL type in Escherichia coli, whereas SHV predominated in Klebsiella spp. and Enterobacter spp. 

 

Clin Microbiol InfectAbstractPneumocystis jirovecii pneumonia (PCP) is a leading cause of morbidity and mortality in immunocompromised patients. Despite the sensitivity of the commonly used PCR for diagnosing P. jirovecii with primers pAZ102-H/pAZ102-E and pAZ102-X/pAZ102-Y derived from mtLSU rRNA (conventional PCR), some PCP patients who had demonstrable organisms by staining methods failed to give positive PCR results. Herein, we devised a more sensitive PCR assay derived from the same gene target to circumvent these false-negative tests. Single brochoalveolar lavage (BAL) samples were collected from human immunodeficiency virus (HIV)-infected (n = 66) and non-HIV (n = 36) immunocompromised patients presenting with fever, dyspnoea, cough and pulmonary infiltrates. Pneumocystis jirovecii was diagnosed with Giemsa-stained smear, immunofluorescence assay, conventional single-round and nested PCR, and new single-round and nested PCR in 46 (45.1%), 53 (52.0%), 69 (67.6%), 74 (72.6%), 87 (85.3%) and 91 (89.2%) patients, respectively. The new PCR could detect P. jirovecii DNA in BAL fluids two to three orders of magnitude more dilute than conventional PCR. Sequence analysis revealed one to three nucleotide substitutions within the primers for conventional PCR among clinical isolates. Although both conventional and new PCR assays were highly specific for diagnosing P. jirovecii, the new PCR yielded more positive results than conventional PCR among BAL samples that were negative by both Giemsa stain and immunofluorescence assay. Hence, the new PCR offered a more sensitive detection of P. jirovecii infection and colonization than conventional PCR. 

 

Clin Microbiol InfectAbstractTo understand the status of oropharyngeal yeast colonization in human immunodeficiency virus (HIV) -infected outpatients in the era of highly active antiretroviral therapy (HAART), we conducted a prospective, cross-sectional study from October 2009 to January 2010 at a medical centre in southern Taiwan. Fungal cultures of the oropharyngeal swabs were performed on 327 enrolled patients. At enrolment, 258 (79%) patients had been receiving HAART, and 42 (12.8%), 73 (22.3%) and 212 (64.8%) patients had CD4 cell counts 200, 201–350, and >350 cells/mm3, respectively. Oral yeast colonization was detected in 193 (59%) patients, among whom 157 (81.3%), 25 (13.0%), and 11 (5.7%) were colonized by a single, two and more than two species, respectively. Multivariate analysis showed that receipt of efavirenz-containing regiments and CD4 cell counts >200 cells/mm3 were associated with lower risks of oral yeast colonization, while intravenous drug users were at a higher risk. Among the 241 isolates recovered, Candida albicans accounted for 69.7%, followed by C. dubliniensis (9.5%), C. glabrata (8.3%), C. tropicalis (3.3%), C. intermedia (2.1%), C. parapsilosis (1.7%), and 11 other species (5.4%). Overall, 230 (95.4%), 236 (97.9%) and 240 (99.6%) isolates were susceptible to fluconazole, voriconazole and amphotericin B, respectively. In conclusion, colonization by C. dubliniensis has emerged in recent years. In addition to a CD4 cell count 200 cells/mm3, which is a known risk factor for oropharyngeal yeast colonization in HIV-infected patients that was identified in our previous studies, two risk factors, non-receipt of efavirenz-based combinations and intravenous drug use, were first identified in the present study. Fluconazole remained effective in vitro against the yeasts colonizing the oropharynx in this population. 

 

Clin Microbiol InfectAbstractTo characterize respiratory virus infections during the first autumn–winter season of pandemic A (H1N1) 2009 influenza virus (A/H1N1/2009) circulation, a prospective study in children attending a paediatric emergency department at the Sapienza University hospital, Rome, was conducted from November 2009 to March 2010. By means of both nasal washings and pharyngeal swabs, enrolled children were checked for 14 respiratory viruses. The majority of acute respiratory infections resulted from viral pathogens (135/231, 58%). Overall, the most common was respiratory syncytial virus (RSV), in 64% of positive samples; A/H1N1/2009 was the only influenza virus found in 16% and rhinovirus (RV) in 15%. Virus-positive children did not differ significantly from virus-negative children in signs and symptoms at presentation; of the virus groups, RSV-infected children were younger and more frequently admitted to intensive-care units than those infected with A/H1N1/2009 and RV. Of the hospitalized children, stratified by age, both infants and children aged >1 year with RSV were most severely affected, whereas A/H1N1/2009 infections were the mildest overall, although with related pulmonary involvement in older children. Children with RV infections, detected in two flares partially overlapping with the A/H1N1/2009 and RSV peaks, presented with bronchiolitis, wheezing and pneumonia. Leukocytosis occurred more frequently in RV-infected and A/H1N1/2009-infected children, and numbers of blood eosinophils were significantly elevated in RV-infected infants. Given the fact that clinical and epidemiological criteria are not sufficient to identify viral respiratory infections, a timely virological diagnosis could allow different infections to be managed separately. 

 

Clin Microbiol InfectAbstractThe presence and characteristics of Clostridium difficile were investigated in 839 faecal samples from seven different animal species in the Netherlands. The number of positive samples ranged from 3.4% (cattle) to 25.0% (dogs). Twenty-two different PCR ribotypes were identified. Among 96 isolates, 53% harboured toxin genes. All C. difficile isolates from pigs, cattle and poultry were toxinogenic, whereas the majority of isolates from pet animals consisted of non-toxinogenic PCR ribotypes 010 and 039. Ribotype 012 was most prevalent in cattle and ribotype 078 in pigs. No predominant ribotypes were present in horse and poultry samples. Overall, PCR ribotypes 012, 014 and 078 were the most frequently recovered toxinogenic ribotypes from animal samples. Comparison with human isolates from the Dutch Reference Laboratory for C. difficile at Leiden University Medical Centre (LUMC) showed that these types were also recovered from human hospitalized patients in 2009/2010, encompassing 0.8%, 11.4% and 9.8% of all isolates, respectively. Application of multiple-locus variable-number tandem-repeat analysis indicated a genotypic relation of animal and human ribotype 078 strains, but a clear genotypic distinction for ribotypes 012 and 014. We conclude that toxinogenic C. difficile PCR ribotypes found in animals correspond to PCR ribotypes associated with human disease in hospitalized patients in the Netherlands. Contrary to PCR ribotype 078, significant genetic differences were observed between animal and human PCR ribotype 012 and 014 isolates. 

 

Clin Microbiol InfectAbstractWe evaluated factors associated with normalization of the absolute CD4+ T-cell counts, per cent CD4+ T cells and CD4+/CD8+ T-cell ratio. A multicentre observational study was carried out in patients with sustained HIV-RNA <50 copies/mL. Outcomes were: CD4-count >500/mm3 and multiple T-cell marker recovery (MTMR), defined as CD4+ T cells >500/mm3plus%CD4 T cells >29%plus CD4+/CD8+ T-cell ratio >1. Kaplan–Meier survival analysis and Cox regression analyses to predict odds for achieving outcomes were performed. Three hundred and fifty-two patients were included and followed-up for a median of 4.1 (IQR 2.1–5.9) years, 270 (76.7%) achieving a CD4+ T-cell count >500 cells/mm3 and 197 (56%) achieving MTMR. Using three separate Cox models for both outcomes we demonstrated that independent predictors were: both absolute CD4+ and CD8+ T-cell counts, %CD4+ T cells, a higher CD4+/CD8+ T-cell ratio, and age. A likelihood-ratio test showed significant improvements in fitness for the prediction of either CD4+ >500/mm3 or MTMR by multivariable analysis when the other immune markers at baseline, besides the absolute CD4+ count alone, were considered. In addition to baseline absolute CD4+ T-cell counts, pretreatment %CD4+ T cells and the CD4+/CD8+ T-cell ratio influence recovery of T-cell markers, and their consideration should influence the decision to start antiretroviral therapy. However, owing to the small sample size, further studies are needed to confirm these results in relation to clinical endpoints. 

 

Clin Microbiol InfectAbstractThe epidemiology and microbiological characteristics of paediatric parapneumonic empyema (PPE) before the introduction of the new generation of conjugate pneumococcal vaccines (10-valent and 13-valent) are described. All patients <14 years old admitted to a tertiary paediatric hospital with a diagnosis of PPE were prospectively enrolled from January 2005 to December 2009. Pneumococcal serotyping of culture-negative pleural fluid samples was performed using a multiplex real-time PCR assay. Overall, 219 patients had PPE. Incidence rates for PPE remained stable during the study period with a not significant increase in 2009 compared with 2005 (p 0.13), and were temporally associated with higher circulation of pandemic influenza A H1N1 during the last quarter in our population (p 0.001). Pneumococci were detected in 72% of culture-positive and 79% of culture-negative samples. Serotypes were determined in 104 PPE cases. Serotype 1 was the most prevalent serotype identified (42%) followed by serotypes 7F (20%), 3 (16%), 19A (8%) and 5 (7%). Serotype distribution remained similar during all time periods. Pneumococcal serotype 1 remained the most common cause of PPE during the 5-year study. The new generation of pneumococcal conjugate vaccines offers potential serotype coverage of 73% (10-valent) and 99% (13-valent) in the population studied suffering from PPE. Continuous epidemiological and molecular studies are paramount to monitor the impact of pneumococcal vaccines on the epidemiology of PPE. 

 

Clin Microbiol InfectAbstractGuidelines state that the CCR5-inhibitor Maraviroc should be prescribed to patients infected with R5-tropic HIV-1 only. Therefore, viral tropism needs to be assessed phenotypically or genotypically. Preliminary clinical trial data suggest that genotypic analysis in triplicate is associated with improved prediction of virological response by increasing the detection of X4-tropic variants. Our objective was to evaluate the impact of triplicate genotypic analysis on prediction of co-receptor usage in routine clinical practice. Samples from therapy-naive and therapy-experienced patients were collected for routine tropism testing at three European clinical centres. Viral RNA was isolated from plasma and proviral DNA from peripheral blood mononuclear cells. Gp120-V3 was amplified in a triplicate nested RT-PCR procedure and sequenced. Co-receptor usage was predicted using the Geno2Pheno[coreceptor] algorithm and analysed with a false-positive rate (FPR) of 5.75%, 10%, or an FPR of 20% and according to the current European guidelines on the clinical management of HIV-1 tropism testing. A total of 266 sequences were obtained from 101 patient samples. Discordance in tropism prediction for the triplicates was observed in ten samples using an FPR of 10%. Triplicate testing resulted in a 16.7% increase in X4-predicted samples and to reclassification from R5 to X4 tropism for four cases rendering these patients ineligible for Maraviroc treatment. In conclusion, triplicate genotypic tropism testing increases X4 tropism detection in individual cases, which may prove to be pivotal when CCR5-inhibitor therapy is applied. 

 

Clin Microbiol InfectAbstractAerococcus urinae is a Gram-positive bacterium that can cause invasive infection, including infectious endocarditis (IE), mainly in older men. A. urinae is often misclassified in routine diagnostic laboratories. Through searches in the laboratory databases we identify 16 isolates of A. urinae causing bacteraemia during a 6-year period in southern Sweden, indicating that bacteraemia with A. urinae occurs in at least three cases per million inhabitants per year. The identity of isolates was confirmed by sequencing of the 16S rRNA genes and antibiotic susceptibility testing identified two ciprofloxacin-resistant isolates. A. urinae was the only significant pathogen isolated in all cases. Fifteen of the 16 patients were male, 15/16 were more than 70 years old, and 12/16 had underlying urological conditions. Though a urinary tract focus was suspected in the majority of cases, the bacterium was rarely found in urinary samples. Nine patients fulfilled the criteria for severe sepsis and an additional four fulfilled the criteria for sepsis. Only one fatality was recorded. Patients were treated mainly with beta-lactam antibiotics but fluoroquinolones and clindamycin were also used. Three cases of IE were diagnosed and these were complicated by spondylodiscitis in one case and by septic embolization to the brain in one case. An increased awareness of A. urinae is crucial to establishing its role as an important pathogen in older men with urinary tract disease. 

 

Clin Microbiol InfectAbstractProsthetic vascular graft infection (PVGI) is a devastating complication, with a mortality rate of up to 75%, which is especially caused by aortic graft infection. The purpose of this study was to evaluate factors associated with in-hospital mortality of patients with definite graft infection, and with long-term outcome. We reviewed medical records of 85 patients treated for PVGIs defined by positive bacterial culture of intraoperative specimens or blood samples, and/or clinical, biological and radiological signs of infection. In-hospital patient mortality was defined as any death occurring during the initial treatment of the graft infection. Cure was defined as the absence of evidence of relapsing infection during long-term follow-up (1 year). Eighty-five patients (54 aortic and 31 limb graft infections) treated by surgical debridement and removal of the infected prosthesis (n = 41), surgical debridement without removal of prosthesis (n = 34) or antimicrobial treatment without surgery (n = 10) were studied. The only microbiological difference observed between patients with early (occurring within 4 months after surgery) vs. late PVGI and between those with aortic vs. limb PVGI was the incidence of PVGI caused by Staphylococcus aureus, which was greater in patients with limb PVGI. Overall cure was observed in 93.2% of 59 patients with a follow-up of a minimum of 1 year. Overall in-hospital mortality was 16.5% (n = 14). Two variables were independently associated with mortality: age >70 years (OR 9.1, 95% CI 1.83–45.43, p 0.007) and aortic graft infection (OR 5.6, 95% CI 1.1–28.7, p 0.037). 

 

Clin Microbiol InfectAbstractImipenem-susceptible E. aerogenes isolates exhibiting extended spectrum ß-lactamases, target mutations and a basal efflux expression, were identified in five patients. After imipenem treatment, imipenem-intermediate susceptible (IMI-I) or resistant (IMI-R) isolates emerged in these patients. Alteration in porin synthesis and increase in efflux expression were observed in the IMI-I isolates whereas complete loss of the porins, LPS alteration and efflux overexpression were observed in the IMI-R isolates. Bacterial virulence of the strains was investigated by the Caenorhabditis elegans model. The IMI-R isolates were shown to be significantly less virulent than the IMI-susceptible or IMI-I isolates. The pleiotropic membrane alteration and its associated fitness burden exhibited by E. aerogenes isolates influence their antibiotic resistance and their virulence behaviour. These findings highlight the balance between the low permeability-related resistance and virulence and their relationships with the treatment of resistant pathogens. 

 

Clin Microbiol InfectAbstractDespite intensive eradication therapy, some CF patients with early Pseudomonas aeruginosa infection rapidly develop a chronic infection. To elucidate factors associated with this persistence, bacterial characteristics of early P. aeruginosa isolates were analysed that were either eradicated rapidly or persisted despite multiple antimicrobial treatments. Eighty-six early infection episodes were studied. First P. aeruginosa isolates from patients with eradication (36) or persistent infection (16) were included; isolates from patients with intermittent infection (34) were omitted from the study. Virulence assays, antimicrobial resistance, cytotoxicity and mutation frequencies were analysed in vitro. P. aeruginosa was genotyped by SNP-array. Transcriptomic profiles of two eradicated and two persistent strains were compared. Nineteen per cent of patients developed persistent infection; 42% achieved eradication. Secretion of virulence factors and mutation frequencies were highly variable among both eradicated and persistent isolates and were not different between the groups. Cytotoxicity was present in 57% of eradicated vs. 100% of persistent isolates (p <0.01). None of the isolates were resistant to antibiotics. The isolates were genotypically highly diverse. Multivariate analysis showed that in vitro determined bacterial characteristics could not predict persistence after first P. aeruginosa infection. Preliminary transcriptomic data showed increased expression of some genes related to a metabolic pathway. The early onset of chronic infection was not associated with (in vitro determined) bacterial characteristics only. Although the persistent isolates were more often cytotoxic, for the individual patient it was not possible to predict the risk of persistence based on bacterial characteristics. Unknown factors such as host-pathogen and pathogen-pathogen interactions should be further explored. 

 

Clin Microbiol InfectAbstractSevere invasive group A streptococcal diseases have re-emerged during the past 10–20 years. In order to provide a better insight into the current epidemiological situation in France, we analysed the questionnaires regarding all invasive strains received at the National Reference Center for Streptococci (CNR-Strep) between 2006 and 2010 from patients aged 18 and characterized them by emm typing, spe gene detection and antibiotic resistance. Among the 1542 invasive GAS strains studied, 78% (n = 1206) were from blood cultures, and a streptococcal toxic shock syndrome (STSS) was described in 22% (n = 340) of cases, mainly associated with necrotizing fasciitis (NF) and pleuro-pulmonary infections (p <0.001). The in-hospital fatality rate was 15%. A total of 83 different emm types were recovered but the three predominant emm types, representing almost 60% of the isolates, were emm1 (24%), emm28 (17%) and emm89 (15%). The preponderance of each emm type varied according to the year, with a significant constant increase of emm28 strains, whereas emm1 strains, representing approximately 32% of GAS invasive isolates in 2007 and 2008, dropped to <15% in 2010 (p <0.001). The distribution of phage-associated superantigen genes (speA, speC and ssa) was linked to certain emm types. Between 2006 and 2010, the percentage that was macrolide-resistant decreased from 11% to 5%, confirming the trend observed in 2007. Fortunately, emm1 strains associated with the most life-threatening clinical manifestations remain susceptible to all anti-streptococcal antibiotics. 

 

Clin Microbiol InfectAbstractTick-borne relapsing fever (TBRF) is a spirochetal infection caused by the genus Borrelia. The disease is distributed in the Old and New World with many different species reported. In Europe, TBRF is caused by B. hispanica transmitted to man by Ornithodoros erraticus, a soft tick usually found in old premises to shelter pig herds. In Portugal, the first human case of TBRF was reported in 1942 but since the beginning of the 1960s, the disease has rarely been described and seems to either have disappeared or have been undiagnosed. Therefore, in 2009 a survey was undertaken to evaluate the presence of the tick in this type of premises and to evaluate its role as a reservoir of Borrelia. The work was carried out where the ticks were previously reported in the Alentejo and Algarve regions. Of 63 pigpens surveyed, O. erraticus was collected from 19% (n = 12) of these pigpens using CO2 traps. To evaluate potential Borrelia hosts, both pigs (n = 25) and small rodents (n = 10) inhabiting these pigpens were surveyed for Borrelia presence, by whole blood PCR and/or tissue culture, respectively. All results for pigs and rodents were negative for the presence of B. hispanica. PCR assays targeting the 16S rRNA gene and intergenic spacer region of Borrelia were used. Sequence analysis of the positive samples confirmed the presence of B. hispanica in 2.2% (n = 5) of ticks from a pigpen in Alentejo. These results confirm natural, but albeit low, persistence of this agent in Portugal. 

 

Clin Microbiol InfectAbstractPhage lysin was evaluated as a substitute for antibiotics in sputum samples processed by a modified Petroff's method for the detection of Mycobacterium tuberculosis with the MGIT 960 system. One hundred and fifty sputum samples were processed, inoculated onto two slopes of Lowenstein–Jensen medium, and divided in to two aliquots of 0.5 mL each. One aliquot was added to 7 mL of MGIT medium containing polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin (PANTA) (MGIT-PANTA) and the other was added to 7 mL of MGIT medium containing 0.8 mL of lysin (MGIT-Lysin). The samples were randomized and incubated at 37°C in the MGIT 960 system. The sensitivity and specificity of MGIT-Lysin were 97% and 88%, respectively, as compared with MGIT-PANTA. The average times to detection with MGIT-Lysin and MGIT-PANTA were 9.3 and 8.6 days, respectively. The rate of contamination with MGIT-PANTA and MGIT-Lysin were 16% and 7.3%, respectively. Phage lysin can be substituted for antibiotics in processed sputum samples for the detection of M. tuberculosis. 

 

Clin Microbiol InfectAbstractAccurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer’s recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory. 

 

Clin Microbiol InfectAbstractPneumococcal surface proteins (PSPs) elicit antibody responses in infants and young children exposed to Streptococcus pneumoniae. These seroresponses could contribute to the aetiological diagnosis of pneumococcal disease, e.g. during the clinical development of novel PSP-based vaccines. In this study, we assessed the kinetics of antibody responses to three highly conserved and immunogenic PSPs (pneumococcal histidine triad D (PhtD), pneumococcal choline-binding protein A (PcpA), and serine proteinase precursor A (PrtA)) in 106 children (median age, 21.3 months; males, 58.5%) admitted for pneumococcal bacteraemia. Anti-PhtD, anti-PcpA and anti-PrtA antibodies were measured by ELISA, and compared in 61 pairs of acute (7 days) and convalescent (>14 days of admission) serum samples. Acute serum titres were similar to those observed in healthy children, and were unaffected by the acid dissociation of circulating immune complexes. Despite proven bacteraemia, seroresponses (2-fold increase in anti-PSP antibody concentrations) were only identified in 31 of 61 children (50.8%), directed against PrtA (n = 23, 37.7%), PcpA (n = 19, 31.1%), and PhtD (n = 16, 26.2%), or several PSPs (two PSPs, n = 13, 21.3%; three PSPs, n = 7, 11.5%). Certain seroresponses were very strong (maximal fold-increases: PhtD, 26; PcpA, 72; PrtA, 12). However, anti-PSP antibody concentrations failed to increase in the convalescent sera of 30 of 61 (49.2%) bacteraemic children, and even declined (2 fold) in 13 of 61 (21.3%), mostly infants aged <6 months (8/13, 61.5%), possibly through consumption of maternal antibodies. Thus, pneumococcal bacteraemia may fail to elicit antibody responses, and may even have an antibody-depleting effect in infants. This novel observation identifies an important limitation of serology-based studies for the identification of bacteraemic children. 

 

Clin Microbiol InfectAbstractThe measurement of early bactericidal activity (EBA) is the first step in the clinical investigation of antituberculosis agents. EBA is determined by quantifying the viable sputum mycobacterial load on consecutive days of treatment. To investigate whether time to positivity (TTP) in mycobacterial liquid culture can substitute for colony forming unit (CFU) counting on agar plates we compared the error variation of TTP and CFU in 2115 pooled sputum samples collected overnight from 250 individuals included in five EBA studies. We found that the technical variation between duplicate laboratory measurements and the within-subject or day-to-day variation were similar for TTP (8.5% and 27.4% of total variation, respectively) and CFU (6.7% and 29.3% of total variation). The ability of the measurements to separate the EBA of 22 treatment arms was determined with group rank correlation of means and one-way analysis of variance. Except for the EBA over 0–2 days, individual and group EBAs measured with TTP and CFU were highly correlated. Treatment group means rank correlation coefficients were r = 0.472, r = 0.910 and r = 0.818, respectively, for EBA 0–2 days, EBA 0–7 days and EBA 0–14 days. Analysis of variance significantly favoured TTP over CFU for discrimination between groups with F values of 6.58 and 1.87, 7.77 and 4.58, and 8.71 and 3.56, respectively. We conclude that TTP is an acceptable alternative to CFU counting for the determination of the viable sputum mycobacterial load in EBA studies of up to 14 days duration. 

 

Clin Microbiol InfectAbstractTraumatic brain injury (TBI) victims are considered to be at high risk for infection. The purpose of this cohort study was to delineate the rates, types and risk factors for infection in TBI patients. Retrospective surveillance of infections was conducted for all TBI patients, aged 18 years, cared for at the Department of Neurosurgery of the University Hospital of Heraklion, Greece, between 1999 and 2005. A total of 760 patients (75% men) with a median age of 41 years were included. Most (59%) were injured in a motor vehicle accident. One third of them underwent a surgical procedure. Two hundred and fourteen infections were observed. The majority were infections of the lower respiratory tract (47%), followed by surgical site infections (SSI) (17%). Multivariate analysis showed that SSI development was independently associated with the performance of 2 surgical procedures (OR 16.7), presence of concomitant infections, namely VAP (OR 5.7) and UTI (OR 8.8), insertion of lumbar (OR 34.5) and ventricular drains (OR 4.0), and cerebrospinal fluid (CSF) leak (OR 3.8). Development of meningitis was associated with prolonged hospitalization (OR 1.02), especially >7 days ICU stay (OR 25.5), and insertion of lumbar (OR 297) and ventricular drains (OR 9.1). There was a notable predominance of Acinetobacter spp. as a VAP pathogen; gram-positive organisms remained the most prevalent in SSI cases. Respiratory tract infections were the most common among TBI patients. Device-related communication of the CSF with the environment and prolonged hospitalization, especially in the ICU setting, were independent risk factors for SSIs and meningitis cases. 

 

Clin Microbiol InfectAbstractFew comprehensive studies have searched for viruses and bacteria in children with community-acquired pneumonia (CAP). We identified 76 children hospitalized for pneumonia. Induced sputum samples were analysed for 18 viruses by antigen detection and PCR, and for six bacteria by culture and PCR. Viruses were found in 72% of samples, bacteria in 91%, and both in 66%. Rhinovirus (30%), human bocavirus (18%) and human metapneumovirus (14%) were the most commonly detected viruses. Two viruses were found in 22% of samples and three in 8%. The most common bacteria found were Streptococcus pneumoniae (50%), Haemophilus influenzae (38%), and Moraxella catarrhalis (28%). Rhinovirus–S. pneumoniae was the most commonly found combination of virus and bacterium (16%). All six children with treatment failure had both viruses and bacteria detected in the sputum. Otherwise, we found no special clinical characteristics in those with mixed viral–bacterial detections. With modern molecular diagnostic techniques, there are high rates of both viral and bacterial identification in childhood CAP. The clinical significance of mixed viral–bacterial infections remains unclear, although we found a potential association between them and treatment failure. 

 

Clin Microbiol InfectAbstractTo assess potential differences in epidemiology and management of patients admitted with influenza infection in the intensive care unit (ICU) during the first post-pandemic influenza period. Observational prospective study comparing September 2009–January 2010 with September 2010–January 2011. Variables captured: demographics, co-morbidities, physiological parameters, outcomes and management. Analysis was performed using SPSS v. 13.0; significance was set at p 0.5. Data from 53 patients, 38 adults (age, median 41.5 years; interquartile range (IQR) 32.8–51.3) and 15 children (age, median 2 years, IQR 0.5–9) are presented. Vaccination rates were 0% and 4.3% during the first and second periods, respectively. Differences postpandemic were: 100% of episodes developed after December compared with 16.7% in the 2009 season. Younger children were affected (median age 0.8 years (IQR 0.3–4.8) vs 7 years (IQR 1.25–11.5), p 0.05) and influenza B caused 8.7% of ICU admissions. Influenza A (H1N1) 2009 and respiratory syncytial virus epidemics occurred simultaneously (42.8% of children) and bacterial co-infections doubled (from 10% to 21.7%); the prevalence of co-infections (viral or bacterial) increased from 10% to 39.1% (OR 5.8, 95% CI 1.3–24.8). Respiratory syndromes without chest X-ray opacities reflecting exacerbation of asthma or chronic obstructive pulmonary disease, bronchitis or bronchiolitis increased (from 6.9% to 39.1%, p <0.05) and pneumonia decreased (from 83.3% to 56.5%, p <0.05). Primary viral pneumonia predominated among ICU admissions. Postpandemic ICU influenza developed later, with some cases of influenza B, more frequent bacterial and viral co-infections and more patients with severe acute respiratory infection with normal chest X-ray. Increasing vaccination rates among risk-group individuals is warranted to prevent ICU admission and death. 

 

Clin Microbiol InfectAbstractListeriosis is a resurgent foodborne disease in European countries. Benefits of combined ß-lactam-aminoglycoside treatment remain controversial and the impact of the underlying disease on prognosis has not been fully assessed. We conducted a retrospective review of cases of sporadic listeriosis in adults from 1995 to 2008 at two university-affiliated hospitals serving a population of 600 000 people in Madrid, Spain. The primary end-point was the associated in-hospital mortality. Sixty-four patients were studied. Estimated incidence of listeriosis was 0.76/100.000 persons/year. Seventy-four per cent had chronic underlying diseases; cirrhosis of the liver and haematological and solid neoplasias were the most common comorbidities. Primary bacteraemia (58%) and meningitis (42%) were the most frequent manifestations. Focal infections were seen in ten cases. In-hospital mortality was 31%. Patients treated with ampicillin or with an ampicillin-gentamicin combination did not differ in age, severity of underlying disease or type of presentation. Differences in mortality were not seen between patients treated with monotherapy and those given combined treatment (28% vs 35%; p 0.634). Ten patients were treated with trimethoprim-sulfamethozaxole alone and only one died. All patients without comorbidities survived infection but mortality of patients with cirrhosis of the liver was 21% and that of patients with haematological or solid neoplasias was 66%. Only haematological neoplasia (OR 6.67; 95% CI 1.71–26.04; p 0.006) was significantly associated with an increased risk of mortality (R2Cox–Snell = 0.262). Mortality of listeriosis mainly depended on the severity of the underlying disease. Combined ampicillin-gentamicin therapy did not improved survival. Trimethoprim-sulfamethozaxole may be an effective alternative therapy for listerial infections. 

 

Clin Microbiol InfectAbstractPersistent Staphylococcus aureus nasal carriers are at high risk of S. aureus infection. The present study delineates a simple strategy aimed at identifying rapidly and accurately this subset of subjects for clinical or epidemiological purposes. Ninety healthy volunteers were each identified as persistent, intermittent or non-nasal carriers of S. aureus by using seven specimens sampled over a 5-week period. By reference to this so-called reference standard, six other strategies aimed at simplifying and speeding the identification of persistent carriers and based on the qualitative or quantitative detection of S. aureus in one to three nasal samples were evaluated by the measure of the area under the curve of receiver operating characteristic diagrams. Among strategies using qualitative results, there was no statistical difference between protocols using seven and three samples. A threshold of 103 CFU of S. aureus per swab was found capable of defining persistent nasal carriage with a sensitivity of 83.1% and a specificity of 95.6%. These figures reached 95.5% and 94.9%, respectively, by using an algorithm including one or two nasal specimens according to the threshold of 103 CFU of S. aureus in the first swab. The latter two strategies were shown to be costly equivalents. The proposed algorithm-based strategy proved to be relevant to identify properly and consistently persistent nasal carriers of S. aureus. However, as it was built from data of healthy volunteers, it needs to be confirmed prospectively on patients potentially at risk for S. aureus infection. 

 

Clin Microbiol InfectAbstractMultidrug-resistant Gram-negative bacteria (MDR-GNB) are an emerging public health threat. Accurate estimates of their clinical impact are vital for justifying interventions directed towards preventing or managing infections caused by these pathogens. A retrospective observational cohort study was conducted between 1 January 2007 and 31 July 2009, involving subjects with healthcare-associated and nosocomial Gram-negative bacteraemia at two large Singaporean hospitals. Outcomes studied were mortality and length of stay post-onset of bacteraemia in survivors (LOS). There were 675 subjects (301 with MDR-GNB) matching study inclusion criteria. On multivariate analysis, multidrug resistance was not associated with 30-day mortality, but it was independently associated with longer LOS in survivors (coefficient, 0.34; 95% CI, 0.21–0.48; p < 0.001). The excess LOS attributable to multidrug resistance after adjustment for confounders was 6.1 days. Other independent risk factors for higher mortality included male gender, higher APACHE II score, higher Charlson comorbidity index, intensive care unit stay and presence of concomitant pneumonia. Concomitant urinary tract infection and admission to a surgical discipline were associated with lower risk of mortality. Appropriate empirical antibiotic therapy was neither associated with 30-day mortality nor LOS, although the study was not powered to assess this covariate adequately. Our study adds to existing evidence that multidrug resistance per se is not associated with higher mortality when effective antibiotics are used for definitive therapy. However, its association with longer hospitalization justifies the use of control efforts. 

 

Clin Microbiol InfectAbstractThe aim of this study was to investigate travel-associated morbidity in European travellers in 2009 in comparison with 2008, with a particular emphasis on emerging infectious diseases with the potential for introduction into Europe. Diagnoses with demographic, clinical and travel-related predictors of disease from ill returning travelers presenting to 12 core EuroTravNet sites from January to December 2009 were analysed. A total of 6392 patients were seen at EuroTravNet core sites in 2009, as compared with 6957 in 2008. As compared with 2008, there was a marked increase in the number of travellers exposed in North America and western Europe. Respiratory illnesses, in particular pandemic A(H1N1) influenza, influenza-like syndromes, and tuberculosis, were also observed more frequently. A significant increase in reported dengue cases in 2009 as compared with 2008 was observed (n = 172, 2.7% vs. n = 131, 1.90%) (p 0.002). The numbers of malaria and chikungunya cases were also increasing, although not significantly. Two deaths were recorded: visceral leishmaniasis and sepsis in a Sudanese migrant, and Acinetobacter sp. pneumonia in a patient who had visited Spain. This is the most comprehensive study of travel-related illness in Europe in 2009 as compared with 2008. A significant increase in travel-related respiratory and vector-borne infections was observed, highlighting the potential risk for introduction of these diseases into Europe, where competent vectors are present. The number of traveller deaths is probably underestimated. The possible role of the travellers in the emergence of infectious diseases of public health concern is highlighted. 

 

Clin Microbiol InfectAbstractDiagnosis of latent tuberculosis infection is a myth for want of a simple, direct tool. Simulation of hypoxic environment was done to create a novel hypothetical model for persistence using processed sputum samples. The adaptation of tubercle bacilli to hypoxic environment seems to be influenced by pre-existing clinical status of the patients at the time of sputum collection, resulting in varied growth pattern. Bacilli from 36 samples did not get adapted to latency of which 15 samples were from patients in whom the disease was well established and the tubercle bacilli in them probably did not experience any stress whatsoever. Similarly, 10 of the 37 samples showing the presence of cultivable cells in both aerobic and anaerobic conditions were from patients who had relapsed. The bacilli in these samples had been probably experiencing stress and thus were ready to adapt to the hypoxic environment. Diagnostic luciferase reporter phage assay for non-replicating persistors (DLRPA-NRP) identified 30 additional positives which failed to grow on Lowenstein–Jensen medium. Presence of viable bacilli in these samples was confirmed by reverse transcriptase-PCR (RT-PCR) for 16S rRNA indicating either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistors. The utility of LRP assay to detect both dormant and active tubercle bacilli was explored in this work and was optimized using lysis inhibition to diagnose tuberculosis with rapidity, improved sensitivity and specificity. DLRPA-NRP, a rapid growth based assay is thus developed to detect both dormant and actively growing tubercle bacilli. 

 

Clin Microbiol InfectAbstractEnterococcus is an important cause of bacteraemia. Previous epidemiological studies examining risk factors for enterococcal bacteraemia have used traditional case–control study designs, which can be potentially biased. This case–case–control study examining risk factors for enterococcal bacteraemia was conducted over 10 years (January 2000 to December 2009) in a tertiary, university-affiliated hospital. There were 440 episodes of enterococcal bacteraemia, 80 of which were caused by vancomycin-resistant Enterococcus (VRE). Two multivariable models were generated, comparing VRE and vancomycin-susceptible Enterococcus (VSE) with the same control group. VRE bacteraemia was associated with central venous catheter use (OR 11.6, 95% CI 2.6–51.5), neutropenia (OR 16.9, 95% CI 2.4–120.2), and allogenic bone marrow transplantation (OR 18.0, 95% CI 2.4–133.4). In contrast, VSE bacteraemia risk factors included: age (OR 1.0, 95% CI 1.0–1.1), exposure to metronidazole (OR 8.7, 95% CI 1.7–43.5), and gastrointestinal disease (OR 6.4, 95% CI 1.2–34.5). Meropenem use decreased the risk of VSE bacteraemia (OR 0.3, 95% CI 0.1–0.9). Hypoalbuminaemia was the only factor identified in both models (VRE, OR 6.0, 95% CI 1.7–21.1; VSE, OR 3.3, 95% CI 1.4–7.7). The absence of substantial overlap of risk factors for VRE and VSE argues in favour of differences in pathogenesis. These data suggest that environmental sources are more important in VRE bacteraemia. Endogenous sources, particularly the gastrointestinal tract, play a pivotal role in VSE bacteraemia. This study highlights the importance of infection control protocols to reduce the risk of VRE bacteraemia. 

 

Clin Microbiol InfectAbstractMany different definitions for multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria are being used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria. A group of international experts came together through a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), to create a standardized international terminology with which to describe acquired resistance profiles in Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae (other than Salmonella and Shigella), Pseudomonas aeruginosa and Acinetobacter spp., all bacteria often responsible for healthcare-associated infections and prone to multidrug resistance. Epidemiologically significant antimicrobial categories were constructed for each bacterium. Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA). MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR was defined as non-susceptibility to all agents in all antimicrobial categories. To ensure correct application of these definitions, bacterial isolates should be tested against all or nearly all of the antimicrobial agents within the antimicrobial categories and selective reporting and suppression of results should be avoided. 

 

Clin Microbiol InfectAbstractThe possibility of using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of pathogenic and non-pathogenic species of the genus Prototheca has been recently demonstrated. A unique reference database of MALDI-TOF MS profiles for type and reference strains of the six generally accepted Prototheca species was established. The database quality was reinforced after the acquisition of 27 spectra for selected Prototheca strains, with three biological and technical replicates for each of 18 type and reference strains of Prototheca and four strains of Chlorella. This provides reproducible and unique spectra covering a wide m/z range (2000–20 000 Da) for each of the strains used in the present study. The reproducibility of the spectra was further confirmed by employing composite correlation index calculation and main spectra library (MSP) dendrogram creation, available with MALDI Biotyper software. The MSP dendrograms obtained were comparable with the 18S rDNA sequence-based dendrograms. These reference spectra were successfully added to the Bruker database, and the efficiency of identification was evaluated by cross-reference-based and unknown Prototheca identification. It is proposed that the addition of further strains would reinforce the reference spectra library for rapid identification of Prototheca strains to the genus and species/genotype level. 

 

Clin Microbiol InfectAbstractThe duration of antimicrobial therapy after surgery for infective endocarditis (IE) is controversial. A short course of postsurgical therapy is currently accepted only for patients with negative valve culture. We performed a retrospective (1994–2008) analysis of patients who underwent surgery for IE in our hospital and had a high risk of complications ( one of more of the following: <2 weeks of antibiotic treatment before surgery; embolism; perivalvular extension; and positive valve culture) to compare outcomes of patients who received short-course antimicrobial therapy (SAT) (median 15 days) or long-course antimicrobial therapy (LAT) (median 32 days), irrespective of the results of valve culture. Our endpoints included length of hospital stay, renal and hepatic failure, relapse, re-infection, and mortality rates 1 year after surgery. During the study period, 140 patients underwent surgery for IE (valve replacement, 87.9%). Of these, 133 fulfilled the high-risk group criteria and 92 completed the antimicrobial schedule. Comparison of patients receiving SAT (37) and LAT (55) showed that the SAT group had a shorter length of hospital stay (29 vs. 40 days, p 0.01), and a trend towards lower frequency of renal failure (5.4% vs. 18.2%, p 0.11) and hepatic failure (5.4% vs. 9.1%, p 0.69), whereas mortality (5.4% vs. 3.6%, p 1), relapse (0% vs. 1.8%, p 1) and re-infection (5.4% vs. 3.6%, p 1) rates were similar between both groups. Multivariate analysis showed that IE caused by Streptococcus viridans or Streptococcus bovis was independently associated with SAT. Postsurgical SAT is safe, especially when IE is caused by Streptococcus viridans or Streptococcus bovis, even in patients at high risk of complications. 

 

Clin Microbiol InfectAbstractTyphoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum ß-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC  4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever. 

 

Clin Microbiol InfectAbstractAt present, colistin is among the few antibiotics effective against Acinetobacter baumannii clinical isolates. However, in the last few years, colistin-resistant A. baumannii strains have been isolated. Therefore, antibiotics effective against these usually pan-resistant colistin-resistant A. baumannii strains are required. The main objective of this study was to analyse the activity of 15 peptides against colistin-susceptible and colistin-resistant A. baumannii. The MICs were determined by microdilution. Among these 15 antimicrobial peptides (AMPs), melittin, indolicidin and mastoparan showed good activity against both colistin-susceptible and colistin-resistant A. baumannii. Further studies of mastoparan with time-killing curves showed bactericidal activity at MIC ×8 for both colistin-susceptible and colistin-resistant A. baumannii. In conclusion, mastoparan may be a potential alternative for the treatment of colistin-resistant A. baumannii infections. 

 

Clin Microbiol InfectAbstractAlthough Clostridium difficile (C. difficile) is the leading cause of infectious diarrhoea in hospitalized patients, the economic burden of this major nosocomial pathogen for hospitals, third-party payers and society remains unclear. We developed an economic computer simulation model to determine the costs attributable to healthcare-acquired C. difficile infection (CDI) from the hospital, third-party payer and societal perspectives. Sensitivity analyses explored the effects of varying the cost of hospitalization, C. difficile-attributable length of stay, and the probability of initial and secondary recurrences. The median cost of a case ranged from $9179 to $11 456 from the hospital perspective, $8932 to $11 679 from the third-party payor perspective, and $13 310 to $16 464 from the societal perspective. Most of the costs incurred were accrued during a patient’s primary CDI episode. Hospitals with an incidence of 4.1 CDI cases per 100 000 discharges would incur costs $3.2 million (hospital perspective); an incidence of 10.5 would lead to costs $30.6 million. Our model suggests that the annual US economic burden of CDI would be $496 million (hospital perspective), $547 million (third-party payer perspective) and $796 million (societal perspective). Our results show that C. difficile infection is indeed costly, not only to third-party payers and the hospital, but to society as well. These results are consistent with current literature citing C. difficile as a costly disease. 

 

Clin Microbiol InfectAbstractJapanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology. 

 

Clin Microbiol Infect 2011; 00: 000–000AbstractA total of 3160 clinical isolates of Escherichia coli from intra-abdominal infections were collected during 2008–2009 from 13 European countries. The frequency of extended-spectrum ß-lactamase (ESBL)-producing isolates in Europe was 11%. The most active antibiotics tested were typically imipenem, ertapenem, and amikacin, although the activity of all non-carbapenem antibiotics was lower when tested against ESBL-positive isolates than when tested against ESBL-negative isolates. Ertapenem exhibited 99.3% susceptibility with all isolates, and 96.8% susceptibility with ESBL-positive isolates. With application of the ertapenem CLSI clinical breakpoint for resistance (MIC 1 mg/L), only six isolates (0.2%) were ertapenem-resistant, and only three of these were available for molecular characterization. Of those three, only one was ESBL-positive (CTX-M-14), and two were carbapenemase-positive (OXA-48). All three were negative for, VIM, NDM and KPC carbapenemases. Although the level of ertapenem resistance in E. coli is very low, further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates is needed. 

 

Clin Microbiol InfectAbstractUnlike tuberculous peritonitis, peritonitis due to non-tuberculous mycobacteria (NTM) has unclear clinical manifestations. This study aimed to clarify the clinical manifestations and laboratory results of NTM peritonitis and compare it to tuberculous peritonitis. This retrospective study was conducted from 2000 to 2008 in a medical centre in Taiwan. Patients with mycobacteria isolated from ascites were identified and compared according to causative pathogens (Mycobacterium tuberculosis or NTM). Those with NTM peritonitis were further classified into the probable’ and possible’ groups based on diagnostic evidence. Twenty-five patients with NTM peritonitis and 65 with tuberculous peritonitis were reviewed. Mycobacterium avium complex was the most common NTM pathogen (52%). There was no obvious difference between the probable’ and possible’ NTM peritonitis groups regarding age and laboratory data. Patients with NTM peritonitis and those with tuberculous peritonitis had no differences in age or gender but varied in symptoms and serum laboratory data. NTM peritonitis was 100% associated with underlying co-morbidities and had lower proportions of lymphocytes and albumin level in ascites. Twelve (48%) NTM peritonitis and 21 (32%) tuberculous peritonitis patients died during the 6-month follow-up. Anti-mycobacterial treatment, but not mycobacterial species, was correlated with better 6-month survival. In Taiwan, NTM is responsible for 28% of mycobacterial peritonitis cases, which have a poor prognosis if untreated. There are some differences in clinical manifestations between NTM and tuberculous peritonitis. NTM peritonitis should be considered in patients with peritonitis but without causative microorganisms identified other than NTM. 

 

Clin Microbiol Infect 2011AbstractFew cases of bacterial meningitis during pregnancy have been reported in the literature, and the causative microorganisms and prognosis of bacterial meningitis during pregnancy are unclear. In a 6-year period we identified six cases of bacterial meningitis in pregnant women. All were multigravida and gestational age at presentation ranged from 5 to 39 weeks. Predisposing factors were present in five patients and consisted of otitis in four patients. The causative organism was Streptococcus pneumoniae in all patients. Two patients died, both due to florid septic shock and brain herniation. Foetal outcome was good in five cases; one woman had a miscarriage 3 weeks after the episode of bacterial meningitis. We reviewed the literature on bacterial meningitis during pregnancy and identified 42 cases of bacterial meningitis. Twenty-five of these patients had pneumococcal meningitis and seven had meningitis caused by L. monocytogenes. We found that pneumococcal meningitis during pregnancy can be rapidly fatal and is associated with foetal death, especially in the first trimester. L. monocytogenes meningitis was associated with a high rate of neonatal deaths. 

 

Since it is unknown whether ß-lactam antimicrobial agents can be used effectively against borderline oxacillin-resistant Staphylococcus aureus (BORSA) with oxacillin MICs 4 mg/L, the in vitro bactericidal activity and pharmacodynamic effect of oxacillin against clinical BORSA isolates was evaluated. Time-kill experiments with oxacillin were performed and the results compared with those obtained with vancomycin, daptomycin and linezolid against BORSA with oxacillin MICs 4 mg/L and BORSA with oxacillin MICs 2 mg/L. Furthermore, the effect of ß-lactamase production and plasmid profile analysis were taken into account to clarify responses to oxacillin. Oxacillin killing activity was attenuated against BORSA compared with ATCC 29213 since the pharmacodynamic parameters revealed that the potency of oxacillin was markedly reduced (c. ten-fold) against BORSA with oxacillin MICs 4 mg/L. pBORa53-like plasmid-containing BORSA with oxacillin MICs 2 mg/L showed markedly more regrowth. In conclusion, oxacillin was non-effective in the eradication of either (i) BORSA with oxacillin MICs 4 mg/L or (ii) ß-lactamase-hyperproducing BORSA (MICs 2 mg/L). Further investigation into ß-lactam dosing strategies against different BORSA strains is warranted in order to avoid possible therapy failure. 

 

A fatal case of disseminated Scopulariopsis brevicaulis infection in an allogeneic stem cell transplant recipient is described. The patient was initially thought to have pulmonary aspergillosis, on the basis of clinical signs and antigenaemia, but Aspergillus was not isolated by culture. Scopulariopsis brevicaulis was subsequently isolated from skin and then from sputum and stool. Further investigation revealed that the infection had spread from a primary pulmonary site to the skin. A review of the literature underscores the difficulty of diagnosing infections caused by such emerging fungal pathogens and the poor outcome of immunocompromised patients with non-Aspergillus mould infections. 

 

The use of alcohol-based hand rubs serves to reduce hospital-acquired infections. Many products of this type are now on offer and it is essential to know how to rank their efficacy. A sequence of tests is proposed here to compare any given new alcohol-based solution against the reference solution (60% 2-isopropyl-alcohol) with 30 s of contact time: (i) in vitro (with pig skin as carrier) testing of >30 species of microorganism; (ii) in vitro assessment of residual efficacy (after 30 min of drying); (iii) in vivo study of transient microbiota (modification of the EN 1500 standard procedure) using four ATCC strains; (iv) in vivo study of resident hand microbiota. After performing the in vitro evaluation of seven alcohol-based hand rubs, the two most efficacious (chlorhexidine-quac-alcohol and mecetronium-alcohol) were chosen and studied, comparatively with the reference solution (60% isopropyl alcohol), in vitro (for chemical sustainability on the skin) and in vivo (against transient and resident microbiota). Chlorhexidine-quac-alcohol proved to be significantly superior to mecetronium-alcohol or the reference solution in all tests, except against resident microbiota for which the improvement was not statistically significant. 

 

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Clin Microbiol Infect 2012; 18: 112–119AbstractDespite the discovery in the last decade of azoles and echinocandins as novel and potent antimycotic drugs, systemic Candida infections are still accompanied by an unacceptably high burden of morbidity and mortality. A rational novel therapeutic approach would be the use of adjuvant immunotherapy, with the aim of improving host defence against Candida. Increases in our understanding of the mechanisms that underlie the pathogenesis of Candida infections, such as the role played by pattern recognition receptors and the induction of proinflammatory cytokines during the early phases of infection, have led to the hypothesis of a potential therapeutic role of recombinant cytokines in systemic candidiasis. In the present review, we give an update of both experimental data and proof-of-principle studies in humans that argue for the use of adjunctive immunotherapy with recombinant cytokines in invasive Candida infections. Sufficiently powered studies on the role of cytokine-based treatment regimens for invasive candidiasis are needed to fully demonstrate the feasibility of this immunotherapeutic approach to improve the prognosis of severe invasive Candida infections. 

 

Clin Microbiol Infect 2012; 18: 120–125AbstractManagement of invasive aspergillosis in high-risk patients remains challenging. There is an increasing demand for novel therapeutic strategies aimed at enhancing or restoring antifungal immunity in immunocompromised patients. In this regard, modulation of specific innate immune functions and vaccination are promising immunotherapeutic strategies. Recent findings have also provided a compelling rationale for assessment of the contribution of the individual genetic profile to the immunotherapy outcome. Altogether, integration of immunological and genetic data may contribute to the optimization of therapeutic strategies exerting control over immune pathways, ultimately improving the management of fungal infections in high-risk settings. 

 

Clin Microbiol Infect 2012; 18: 126–133AbstractDespite appropriate antifungal treatment, the management of cryptococcal disease remains challenging, especially in immunocompromised patients, such as human immunodeficiency virus-infected individuals and solid organ transplant recipients. During the past two decades, our knowledge of host immune responses against Cryptococcus spp. has been greatly advanced, and the role of immunomodulation in augmenting the response to infection has been investigated. In particular, the role of protective’ Th1 (tumour necrosis factor-, interferon (IFN)-, interleukin (IL)-12, and IL-18) and Th17 (IL-23 and IL-17) and non-protective’ Th2 (IL-4, IL-10, and IL-13) cytokines has been extensively studied in vitro and in animal models of cryptococcal infection. Immunomodulation with monoclonal antibodies against the capsular polysaccharide glucuronoxylomannan, glucosylceramides, melanin and ß-glucan and, lately, with radioimmunotherapy has also yielded promising results in animal models. As a balance between sufficiently protective Th1 responses and excessive inflammation is important for optimal outcome, the effect of immunotherapy may range from beneficial to deleterious, depending on factors related to the host, the infecting organism, and the immunomodulatory regimen. Clinical evidence supporting immunomodulation in patients with cryptococcal infection remains too limited to allow firm recommendations. Limited human data suggest a role for IFN-. Identification of surrogate markers characterizing patients’ immunological status could possibly suggest candidate patients for immunotherapy and the type of immunomodulation to be administered. 

 

Clin Microbiol Infect 2012; 18: 134–139AbstractInvasive fungal infections caused by rare filamentous fungi constitute a significant cause of morbidity and mortality in patients with defective immune responses. Despite the advent of new antifungal agents, the problem is escalating as the number of susceptible hosts increases and virulent, more resistant fungal strains emerge. There is evidence that reconstitution of the host immune function is a major contributor to the resolution of these infections. Therapeutic modalities aimed at increasing phagocyte numbers, such as granulocyte transfusions, stimulating the immune response, such as administration of haematopoietic growth factors and other proinflammatory cytokines, or indirectly augmenting immune function have shown promising results in the preclinical setting. Because of the rarity of the infections, multicentre clinical trials are needed to demonstrate the efficacy and safety of the new immunomodulating approaches. 

 

Clin Microbiol Infect 2012; 18: 140–146AbstractA strain of Klebsiella pneumoniae (K1) was isolated from a catheterized patient with a urinary tract infection. The patient was subsequently treated with meropenem, after which K. pneumoniae (K2) was once again isolated from the patient’s urine. Susceptibility testing showed that strain K1 was fully susceptible to carbapenem antibiotics with the exception of ertapenem, to which it exhibited intermediate resistance, whilst K2 was resistant to ertapenem and meropenem. From pulsed-field gel electrophoresis profiling both strains exhibited identical banding patterns. Both contained CTX-M-15, OXA-1, SHV-1 and TEM-1 ß-lactamase genes following PCR analyses. Outer membrane protein analysis demonstrated that K1 and K2 lacked an OMP of c. 40 kDa, with an additional OMP of c. 36 kDa missing from K2. Mutation studies showed that the K2 OMP phenotype could be selected by single-step carbapenem-resistant mutants of K1. Expression of transcriptional activator ramA and efflux pump component gene acrA were up-regulated in both strains by RT-PCR. Neither strain expressed ompK35, but ompK36 was found in both. Sequence analysis revealed gene sequences of ompK35, ompK36 and ramR in both strains; notably, ramR contained a mutation resulting in a premature stop codon. Transconjugation studies demonstrated transfer of a plasmid into E. coli encoding the CTX-M-15, TEM-1 and OXA-1 ß-lactamases. We concluded that the carbapenem-resistant phenotype observed from this patient was attributable to a combination of CTX-M-15 ß-lactamase, up-regulated efflux and altered outer membrane permeability, and that K2 arose from K1 as a direct result of meropenem therapy. 

 

Clin Microbiol Infect 2012; 18: 147–152AbstractIn order to perform a cost-effective search and destroy policy for methicillin-resistant Staphylococcus aureus (MRSA), a quick and reliable typing method is essential. In an area with a high level of animal-related MRSA ST398, pulsed field gel electrophoresis (PFGE) typing and spa-typing are not sufficient to discriminate between co-incidental findings and true transmission of MRSA. This study is the first to retrospectively show the performance of Raman spectroscopy in 16 well-documented outbreaks. We analysed 525 isolates, 286 MRSA ST398 and 239 from other PFGE clusters with Raman spectroscopy. When epidemiologically linked isolates from the outbreaks were analysed with PFGE as the reference standard, Raman spectroscopy correctly identified 97% of cases that were indistinguishable from the index case. With Raman cluster analysis, the most dominant distinction was between MRSA ST398 and other MRSA of human clonal lineages. Within MRSA ST398, 22 different Raman clusters were identified. Raman typing correctly identified an ST398 (spa type t567) outbreak in a hospital setting. No direct correlation was observed between Raman clusters and spa types. We conclude that Raman spectroscopy is a quick and reliable method of MRSA typing, which can be used in outbreak settings and it is comparable to PFGE, with the added advantage that PFGE non-typeable isolates can also be readily typed using the same sample preparation protocol. 

 

Clin Microbiol Infect 2012; 18: 153–159AbstractThe molecular epidemiology of Helicobacter pylori in Africa is poorly documented. From January 2007 to December 2008, we investigated 187 patients with gastric symptoms in one of the main tertiary hospitals in Dakar, Senegal. One hundred and seventeen patients were culture-positive for H. pylori. Polymorphisms in vacA and cagA status were investigated by PCR; the 3-region of cagA was sequenced, and EPIYA motifs were identified. Bacterial heterogeneity within individuals was extensively assessed by using an approach based on vacA and cagA heterogeneity. Fourteen per cent of H. pylori-positive patients displayed evidence of mixed infection, which may affect disease outcome. Patients with multiple vacA alleles were excluded from subsequent analyses. Among the final study population of 105 patients, 29 had gastritis only, 61 had ulcerated lesions, and 15 had suspicion of neoplasia based on endoscopic findings. All cases of suspected neoplasia were histologically confirmed as gastric cancer (GC). The cagA gene was present in 73.3% of isolates. CagA proteins contained zero (3.7%), one (93.9%) or two (2.4%) EPIYA-C segments, and all were western CagA. Most of the isolates possessed presumed high-vacuolization isotypes (s1i1m1 (57.1%) or s1i1m2 (21.9%)). Despite the small number of cases, GC was associated with cagA (p 0.03), two EPIYA-C segments in the C-terminal region of CagA (p 0.03), and the s1 vacA allele (p 0.002). Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of GC in Senegal. 

 

Clin Microbiol Infect 2012; 18: 160–166AbstractThis study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004–2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried blaOXA-58 alone, 21 of 202 also carried blaOXA-23, and 27 of 202 carried blaOXA-23 alone. No isolates were positive for blaOXA-24. Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones. 

 

Clin Microbiol Infect 2012; 18: 167–170AbstractWe describe a 31-year-old immunocompromised patient who developed sepsis and osteomyelitis due to Salmonella enterica subsp. arizonae secondary to exposure to iguana and snakes kept as pets at her home, and review all 23 previously published cases of bone and joint infections due to this organism, for a total of nine children and 15 adults. Eleven of the adults were female (73%), compared with three (33%) of the children (p <0.01). Significant underlying illnesses were present in all 15 adults and in five children (55%, p <0.05); 10 (77%) of the adults were immunosuppressed, compared with one child only (17%) (p <0.05). In ten of the adults the knee was infected (67%), compared with one child only (11%, p <0.01). Antibiotic therapy was prolonged in both adults and children, and in most cases consisted of 4–6 weeks of parenteral treatment. Complete cure and survival was attained in 11 of 15 adults (73%) and all nine children (NS). Optimal antibiotic treatment probably consists of ceftriaxone or a fluoroquinolone, if the organism is susceptible. 

 

Clin Microbiol Infect 2012; 18: 171–176AbstractDengue fever (DF) remains one of the most important emerging infectious diseases. Whereas DF is well recognized in endemic countries, there are indications that the disease is underdiagnosed among travellers to endemic regions. Here, we present the first descriptive survey on cases of travel-acquired DF imported to Denmark diagnosed at the national reference laboratory for dengue virus diagnostics during a 9-year period. In our study, 16 – 46 travel-acquired dengue virus infections were diagnosed per year. DF is mainly imported by adults, mostly men, returning from Southeast Asian countries. The minimum incidence of dengue virus infection among Danish travellers is estimated to be 4.9 per 100 000 travellers. Our results confirm and expand studies from other European countries, and underline the importance of surveillance based on relevant diagnostic analyses. 

 

Clin Microbiol Infect 2012; 18: 177–183AbstractWe looked for evidence of antibodies to the 2009 influenza A/H1N1 pandemic virus in panels of sera from individuals living in metropolitan France, obtained either before, during or after the epidemic, using standard haemagglutination inhibition and microneutralization tests. The difference between seroprevalence values measured in post- and pre-epidemic panels was used as an estimate of seroconversion rate in different age groups (23.4% (0–24 years, age-group 0); 16.5% (25–34); 7.9% (35–44); 7.2% (45–54); 1.6% (55–64); and 3.1% (>65)), confirming that the distribution of cases in different age groups was similar to that of the seasonal H1N1 virus. During the pre-pandemic period low-titre cross-reactive antibodies were present in a large proportion of the population (presumably acquired against seasonal H1N1) whereas cross-reactive antibodies were detected in individuals over the age of 65 years with significantly higher prevalence and serological titres (presumably acquired previously against Spanish flu-related H1N1 strains). Clinical data and analysis of post-pandemic seroprevalence showed that few of these latter patients were infected by the influenza virus during the epidemic. In contrast, the majority of both clinical cases and seroconversions were recorded in the 0–24 age group and a global inverse relationship between prevalence of antibodies to pH1N1 in the pre-pandemic period and rate of seroconversion was observed amongst age groups. Our results emphasize the complex relationships involved in antigenic reactivity to pandemic and seasonal H1N1 viral antigens; hence the difficulty in distinguishing between low-titre specific and cross-reactive antibodies, establishing precise seroprevalence numbers and fully understanding the relationship between previous immunity to seasonal viruses and protection against the novel variant. 

 

Clin Microbiol Infect 2012; 18: 184–188AbstractThe human pathogen xenotropic murine leukaemia virus-related virus (XMRV) has been tentatively associated with prostate cancer and chronic fatigue syndrome. Unfortunately, subsequent studies failed to identify the virus in various clinical settings. To determine whether XMRV circulates in humans and the relationship with its host, we searched for the virus in 124 human immunodeficiency virus-infected patients who might have been exposed to XMRV, might be prone to infection as a result of progressive immunodeficiency, and had not yet been treated with antiretroviral drugs. Using nested PCR and single-step TaqMan real-time PCR, both designed on the XMRV gag gene, we could not find any positive samples. These findings add to the growing amount of scepticism regarding XMRV. 

 

Clin Microbiol Infect 2012; 18: 189–194AbstractInvasive fungal disease (IFD) remains a significant cause of mortality in haematology patients. Management strategies range from empiric therapy to pre-emptive approaches. Prophylaxis with mould-active agents has been evaluated, but the optimal strategy remains unclear. We present here a retrospective analysis of the pre-emptive strategy implemented at our institution. We analysed 348 consecutive neutropenic episodes in 234 patients. The main elements of our pre-emptive strategy included twice-weekly Galactomannan testing and weekly computed tomography (CT) scan. Antifungal prophylaxis usually consisted of fluconazole (400 mg once weekly). Antifungal treatment was started if criteria for IFD (including possible) were fulfilled. Along with the incidence of antifungal treatment and IFD, we also analysed the adherence to the strategy. Adherence was good but suboptimal with 81% of CT scans having been performed at an interval of 10 days or less. Concerning antifungal treatment, in 56 episodes the patient was receiving a mould-active agent as secondary prophylaxis. Antifungal treatment was started during 39% of the remaining episodes. In all, 109 cases of IFD were diagnosed, 51 being probable or proven. Forty-nine patients died before day 100, IFD directly caused or contributed to death in six patients. Two cases of IFD were diagnosed post-mortem and were missed by our pre-emptive strategy. Our pre-emptive strategy is feasible and safe with an acceptable rate of IFD and associated mortality, while avoiding anti-fungal treatment in a significant proportion of patients. The exact role of pre-emptive treatment compared with systematic mould-active prophylaxis can only be determined in a well-designed randomized trial. 

 

Clin Microbiol Infect 2012; 18: 195–201AbstractCandidaemia is associated with high patient mortality. Among those who survive an initial episode of candidaemia, the incidence of recurrent episodes has been incompletely defined. All patients in Iceland with candidaemia in 1980–2008 were identified, and clinical information was reviewed. Episodes of candidaemia in the same patient were considered to be separate if they occurred 1 month apart or were caused by different Candida species. The isolates were genotyped by using PCR fingerprinting, and antifungal susceptibility was determined. During the 29-year period, candidaemia was diagnosed in 307 patients in Iceland, 298 of whom (97.1%) had a single episode. Overall, 206 patients survived >1 month from the first episode and were therefore at risk of recurrence, yielding 1062 patient-years of observation in the survivors. Of those, nine (4.4%) later developed recurrent candidaemia. The median time between recurrences was 6 months (range, <1 month to 14 years). Patients with late recurrences were younger (p 0.012) and more likely to have underlying gastrointestinal diseases than patients with single episodes (55.6% vs. 18.5%, respectively; p 0.017). The recurrences were caused by identical Candida sp. genotypes in two of 13 cases (15%), but by different species or dissimilar genotypes in eight of 13 (62%); isolates were missing in three cases. In conclusion, late recurrent candidaemia was a relatively rare event, and younger patients with gastrointestinal disorders were more prone to recurrent infections. The majority of late recurrences represented re-infections with new Candida strains that were susceptible to common antifungal agents. 

 

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Clin Microbiol Infect 2012; 18: E16–E19AbstractTo estimate endemic areas for Crimean-Congo haemorrhagic fever (CCHF) in Greece, a country-wide seroepidemiological study was conducted, and 1611 human sera were prospectively collected along with data regarding possible risk factors for acquisition of infection, and tested for CCHF virus IgG antibodies by ELISA. The overall seroprevalence was 4.2%, with significant differences among prefectures, ranging from 0 to 27.5%. Multivariate analysis revealed that slaughtering and agricultural activities were significant risk factors for CCHFV seropositivity. The significantly high seroprevalence in specific prefectures, together with the extremely low number of CCHF cases, suggest that this phenomenon might be strain-related. 

 

Clin Microbiol Infect 2012; 18: E20–E23AbstractWe compared the rate and extent of anidulafungin’s and fluconazole’s activity in neutropenic and non-neutropenic mice with Candida albicans invasive candidiasis. In immunocompetent mice, anidulafungin significantly improved survival vs. controls and fluconazole, and significant reductions in (13)-ß-D-glucan and fungal burden were observed. In neutropenic animals, the highest doses of anidulafungin (5 mg/kg) and fluconazole (10 mg/kg) also improved survival and reduced fungal burden. However, there were no differences in survival between these antifungals as anidulafungin’s activity was attenuated in this model. These results demonstrate that the extent of anidulafungin in vivo efficacy may be dependent on host immune status. 

 

Clin Microbiol Infect 2012; 18: E24–E26AbstractWe used molecular techniques to analyse 87 (n = 70 patients) Aspergillus terreus complex isolates, all of which were identified as A. terreus sensu stricto. The antifungal susceptibilities determined with CLSI M38-A2 (and Etest for amphotericin B) and expressed as mg/L for range of MIC/MIC90/geometric mean were as follows: itraconazole, 0.25–2/2/1.097; voriconazole, 0.125–2/2/1.176; posaconazole, 0.25–1/1/0.836; amphotericin B CLSI, 4–32/16/9.689; and Etest, 0.75–64/6/3.106. The MICs for amphotericin B were significantly higher than those found for the triazoles. 

 

Clin Microbiol Infect 2012; 18: E27–E30AbstractIn this retrospective observational study covering 1998 to 2008, 32 patients (mean age: 7.50 years) were identified that had 35 episodes of candidaemia (0.47 cases/1000 hospital discharges). Cancer/allogeneic haematopoietic stem cell transplantation (43%) and congenital malformations/syndromes (21%) were the predominant underlying conditions. Central venous catheterization (90%), a history of antibacterial therapy (69%) and previous bacteraemia (54%) were frequent comorbidities. Candida albicans (46%) was most common, followed by Candida parapsilosis (17%) and Candida glabrata (14%). Resistance was infrequent and limited to non-albicans Candida spp. The 30-day and 100-day mortality rates were 11.4%. 

 

Clin Microbiol Infect 2012; 18: E31–E33AbstractThe nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat. 

 

Clin Microbiol Infect 2012; 18: E34–E36AbstractScreening 155 carbapenem non-susceptible Acinetobacter baumannii strains recovered in Abu Dhabi hospitals identified two metallo-ß-lactamase blaNDM gene-carrying isolates. They were isolated 4 months apart from the urine of a cancer patient previously treated in Egypt, Lebanon and in the United Arab Emirates. They were clonally related and carried the blaNDM-2 gene recently identified in A. baumannii in Egypt and Israel. Sequences surrounding the blaNDM-2 gene showed significant similarities with those associated with blaNDM-1 in Enterobacteriaceae and A. baumannii. Repeated isolation of blaNDM-2-positive A. baumannii in the Middle East raises the possibility of the local emergence and spread of a unique clone.