Clin Microbiol Infect Research Articles

Clinical features of chronic C virus hepatitis in patients with celiac disease

The association between celiac disease (CD) and several liver disorders has long been documented. About 40% of adult celiac patients have been reported to have mild to moderate hypertransaminasemia (up to five times the upper limit of normal) at the time of diagnosis of CD [1, 2]. In addition, CD has been found in roughly 10% of patients with unexplained hypertransaminasemia, and the majority of them have had their liver enzyme levels normalized after one year of following a strict gluten-free diet [3, 4]. In addition, an increased prevalence of primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis among CD patients has been reported [5, 6]. CD might also be linked to very severe liver conditions such as end-stage liver failure and hepatocellular carcinoma [7]. There is also evidence, even contrasting reports, about the association of CD with nonalcoholic steatohepatitis and fatty liver disease [8]. In contrast, no definitive evidence is available about the association between chronic hepatitis C (hepatitis C virus [HCV]) and CD. Fine et al. described a three-fold increase of CD prevalence among HCV patients compared to noninfected celiac individuals [9]. It has also been reported the activation of silent CD during the antiviral treatment for HCV with interferon-α and ribavirin, both alone and in combination [10]. Consequently, a routine serological screening for CD has been proposed in HCV patients before starting antiviral therapy. In case of HCV positivity, the achievement of the histological normalization of the intestinal mucosa after following a gluten-free diet has been advised before starting the therapy [10]. On the contrary, some recent prospective studies have not shown increased prevalence of CD among HCV patients and reported that the link between these two conditions is biased by the route of transmission [11, 12]. Among the 3,725 celiac patients included in the Italian Registry of the Complication of Celiac Disease, we identified 34 individuals (0.91%) that had an HCV chronic hepatitis at the time of diagnosis of CD. For the diagnosis of HCV, we considered the serological positivity of antiHCV antibodies. Some of the patients had the diagnosis made in the early 1980s, when the molecular tests for the detection of the viral antigens were not yet available. The demographic and clinical features of the patients with both CD and HCV with respect to those of patients with CD only are listed in Table 1. The prevalence of HCV among our celiac series is lower than the overall prevalence of HCV among the general population in Italy, matched for age and gender, which is estimated to be around 2% [13]. This finding does not support the hypothesis of a potential correlation between these two disorders. It has been assumed that antiviral therapy with INF-α and ribavirin may precipitate the onset of CD in susceptible individuals, promoting a Th1-specific response in the small intestine [14]. However, in our series, only 12 of the 34 celiac patients with HCV had antiviral therapy before CD diagnosis. Looking at our series, it seems more likely that an overall increased risk of CD in HCV patients exists, due to the predisposition for autoimmune diseases related to Eur J Clin Microbiol Infect Dis (2009) 28:1267–1269 DOI 10.1007/s10096-009-0769-6

Study on blood cultures and bacteria counts in the blood of paratyphoid fever A patients

Paratyphoid fever A (PA) is a systemic infection caused by Salmonella enterica serotype Paratyphi A (SPA) [1]. Although blood cultures are considered to be the ‘gold standard’ for the detection of bloodstream pathogens, there is little information on the controlled clinical comparison of the paired aerobic and anaerobic bottles in the automated continuously monitoring instrument systems, and bacterial counts in the blood of patients with bloodstream infections [2, 3]. Yuxi City is the most severe endemic area of PA in China [4]. In order to determine the factors determining the yields from blood cultures, we have performed broth blood cultures (BBC) by the BacT/ALERT 3D automated blood culture system (bioMerieux, Inc., Durham, NC) and quantitative whole-blood culture (QWBC) for a large series of patients with suspected PA between 1999 and 2008. A total of 13,500 patients admitted with suspected PA to our institutions between June 1999 and June 2008 were studied. The available blood samples were obtained with vacuum system blood collection tubes. A blood culture set included one aerobic bottle (AEB) and one anaerobic bottle (ANB) (bioMerieux), and one QWBC plate (Columbia agar, bioMerieux). A 13-ml quantity of blood from each patient for one test set was considered to be the maximum amount to obtain from a single venipuncture and was distributed as follows: 5 ml to one AEB, 5 ml to one ANB, and 3 ml to one plate (only for 1,000 probable cases of PA). The culture bottles contained 5 ml of blood sample and 40 ml of culture media. Each bottle was monitored with the BacT/ALERT 3D system at 37°C for a standard 5-day protocol or until the instrument signaled a positive result [5]. Any bottle with a positive signal was immediately removed from the instrument, and an aliquot of the blood– broth mixture was taken from the bottle with a sterile needle and swab for subculture on sheep blood nutrient agar and XLD agar (Oxoid, England); the time-to-detection of bacteria causing PA was taken as the incubation time required before the instrument gave a positive signal. All bottles were treated independently until the end of the 7-day incubation and testing period. Both BBC and QWBC were performed for 1,000 probable cases of PA. QWBC were carried out by a pour plate method and the blood bacterial counts were estimated [3]. In brief, 3 ml of blood was drawn aseptically from each patient and injected into a sterile heparinized tube. Three milliliters of heparinized blood was mixed well with 19 ml of molten (50°C) Columbia agar in a sterile Petri dish, allowed to set for solidification, and then incubated at 37°C. After 2 to 4 days, colonies were identified, counted, and recorded as colony-forming units (CFU) per milliliter. The number of SPA bacteria per milliliter of blood was estimated from the number of CFU on each pour plate. The minimum volume of whole blood used for QWBC from any one patient was 3 ml. This gives a lower limit of Eur J Clin Microbiol Infect Dis (2009) 28:1259–1261 DOI 10.1007/s10096-009-0766-9

Highly specific detection of antibodies to tick-borne encephalitis (TBE) virus in humans using a domain III antigen and a sensitive immune complex (IC) ELISA

Background In contrast to most antibodies directed to the envelope glycoprotein of flaviviruses, those to the domain III (ED3) show serotype-specific reactions. Objectives Only few epitopes are located on the ED3 [Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, et al. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 2005; 11:522–30; Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 2005; 437:764–9; Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile virus. J Virol 2006; 80:6982–92], and highly sensitive assays may be required to detect the small number of human antibodies to this domain. Study design We have used a sensitive immune complex (IC) ELISA to detect antibodies to the ED3 of TBE virus [Ludolfs D, Niedrig M, Paweska JT, Schmitz H. Reverse ELISA for the detection of anti-West Nile virus IgG antibodies in humans. Eur J Clin Microbiol Infect Dis 2007; 26:467–73; Emmerich P, Gunther S, Schmitz H. Strain-specific antibody response to Lassa virus in the local population of west Africa. J Clin Virol 2008; 42:40–4]. This assay was compared with two indirect ELISAs using either the ED3 (ED3 ELISA) or whole tissue culture virus (TCV) (TCV ELISA) as source of antigen. Sera of 45 patients with acute TBE infection and of 65 vaccinees were applied to determine the sensitivity of the IC ELISA. Results The IC ELISA detected antibodies in 107 out of 110 samples of TBE patients and vaccinees, 106 of which were also positive with the TCV ELISA. Both tests had a sensitivity of ≥96%. In contrast, the ED3 ELISA had a sensitivity of only 70%. Using samples of 98 West Africans and of 70 Europeans without any contact to TBE virus or TBE virus antigens, the specificity of the IC ELISA was 100% while the specificity of the commercial TCV ELISA varied between 30% with samples of people with acute dengue fever or with yellow fever vaccination and 100% with samples of students from Hamburg without any previous contact to TBE. Conclusion Obviously, the IC ELISA is able to detect human antibodies to small antigens with only few serotype-specific epitopes with high specificity and sensitivity.

Direct Bacterial Profiling by Matrix-Assisted Laser Desorption−Ionization Time-of-Flight Mass Spectrometry for Identification of Pathogenic Neisseria

The present study investigates the suitability of direct bacterial profiling as a tool for the identification and subtyping of pathogenic Neisseria. The genus Neisseria includes two human pathogens, Neisseria meningitidis and Neisseria gonorrhoeae, as well as several nonpathogenic Neisseria species. Here, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling protocol was optimized using a laboratory strain of E. coli DH5alpha to guarantee high quality and reproducible results. Subsequently, mass spectra for both laboratory and clinical strains of N. gonorrhoeae, N. meningitidis, and several nonpathogenic Neisseria species were collected. Significant interspecies differences but little intraspecies diversity were revealed by means of a visual inspection and bioinformatics examination using the MALDI BioTyper software. Cluster analysis successfully separated mass spectra collected from three groups that corresponded to N. gonorrhoeae, N. meningitidis, and nonpathogenic Neisseria isolates. Requiring only one bacterial colony for testing and using a fast and easy measuring protocol, this approach represents a powerful tool for the rapid identification of pathogenic Neisseria and can be adopted for other microorganisms.

Chronic hepatitis E in the immunosuppressed: A new source of trouble?

Chronic hepatitis E virus infection in liver transplant recipients. Haagsma EB, van den Berg AP, Porte RJ, Benne CA, Vennema H, Reimerink JH, Koopmans MP. Hepatitis E virus (HEV) infection is known to run a self-limiting course. Sporadic cases of acute hepatitis due to infection with HEV genotype 3, present in pig populations, are increasingly recognized. Zoonotic transmission seems infrequent. The entity of unexplained chronic hepatitis after liver transplantation has been recognized. Detection of HEV in 2 liver transplant recipients triggered a review of these cases. Freeze-stored sera were available for retrospective analysis. HEV antibodies were determined. For virus detection and identification, a fragment of the gene encoding the major capsid protein (open reading frame 2) was amplified by reverse-transcription polymerase chain reaction and sequenced to identify the genotype. Two months after liver transplantation, case A developed unexplained chronic hepatitis, which developed into cirrhosis. Retransplantation followed 7 years later, after which chronic hepatitis recurred. In retrospect, HEV RNA was present in serum 3 weeks after the first transplantation and remained present afterwards. HEV RNA was also present in retransplant liver tissue. HEV antibodies appeared late after retransplantation. Case B developed unexplained chronic hepatitis 7 years after transplantation. Retransplantation was needed 5 years later, after which no signs of hepatitis recurred. In retrospect, the period of chronic hepatitis up to the retransplantation coincided with HEV RNA in serum. In case B, antibodies developed, the viral load was much lower than in case A, and the virus seemed to be cleared after retransplantation. Genotyping in both cases revealed 2 unique strains of genotype 3. In conclusion, chronic HEV infection may develop in immunosuppressed patients, who may then serve as long-term carriers of the virus. We hypothesize that HEV may be the cause of chronic hepatitis in liver transplant recipients. (c) 2008 AASLD. [Abstract reproduced by permission of Liver Transpl 2008;14:547–553]. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, Cointault O, Esposito L, Abravanel F, Danjoux M, Durand D, Vinel JP, Izopet J, Rostaing L. Hepatitis E virus (HEV) is considered an agent responsible for acute hepatitis that does not progress to chronic hepatitis. We identified 14 cases of acute HEV infection in three patients receiving liver transplants, nine receiving kidney transplants, and two receiving kidney and pancreas transplants. All patients were positive for serum HEV RNA. Chronic hepatitis developed in eight patients, as confirmed by persistently elevated aminotransferase levels, serum HEV RNA, and histologic features of chronic hepatitis. The time from transplantation to diagnosis was significantly shorter and the total counts of lymphocytes and of CD2, CD3, and CD4 T cells were significantly lower in patients in whom chronic disease developed. [Abstract reproduced by permission of N Engl J Med 2008;358:811–817].

An outbreak of Clostridium difficile-associated disease (CDAD) in a German university hospital

We investigated an outbreak of Clostridium difficile (CD)associated disease (CDAD) in a cardiac surgical department that took place between January 25th and April 28th 2007 at Hannover Medical School, Germany. Immediate infection control measures such as changing the surface disinfectant from an alcohol-based glucoprotamine to an oxygen-active disinfectant cleaner and the primary education of staff were proved to be insufficient to control the outbreak. Therefore, a “CD infection control bundle” was created consisting of: (a) implementation of a CDAD outbreak team; (b) education on hand hygiene; (c) the aim of early case finding as soon as clinical symptoms of CDAD were noticed; (d) daily control of microbiological results; (e) suggesting proper antimicrobial therapy of patients on the ward avoiding high-risk substances; (f) reinforcement of currently existing infection control measures, such as including the isolation of CDAD cases in single rooms; (g) and, finally, interim closure of the unit for any new admissions. For the treatment of CDAD, initially, metronidazole and, in the case of failure of initial therapy, oral vancomycin were used. A case–control study was performed. Cases were defined as proposed by the ECDC: (a) onset of diarrhoea ≥48 h after admission or ≥4 weeks after the most recent discharge and (b) positive enzyme-immunoassay (EIA) for CD toxins A or B in stool samples (RIDASCREEN® Clostridium difficile Toxin A/B test, R-Biopharm, Glasgow, United Kingdom) or the culturing of toxin-producing CD (Clostridium difficile-selective media, Oxoid, Hampshire, United Kingdom). The results of Gram strain, colony morphology and biochemical testing with the RapID II ANA System® (Remel, Lenexa, USA) were used for species determination. Cultured strains were examined by pulsed field gel electrophoresis (PFGE) using GelCompar II® software (Applied Maths NV, Sint-Martens-Latern, Belgium) and by polymerase chain reaction (PCR) ribotyping, as described by others [1]. Antimicrobial susceptibility testing was performed by E-test® (AB Biodisk, Solna, Sweden) on agar plates containing vitamin K1, haemin and 5% defibrinated sheep red blood cells (Inverness Medical Deutschland, Cologne, Germany). Breakpoint minimal inhibitory concentrations (MICs) were ≥8 μg/mL for fluoroquinolones (FQ) [2] and ≥4 μg/mL for erythromycin [3]. Severe CDAD was defined as readmission because of relapse, necessity of intensive care because of CDAD, surgical procedure due to CDAD or CDAD contributing to the patient’s death within 30 days after diagnosis [4–6]. Controls were diarrhoeal patients admitted to the same unit during the same time period but were negative for CD toxin EIA, including CD culture (= confirmed CD negative) and negative for other gastrointestinal infectious agents. Age, gender, co-morbidities, underlying diseases, endoscopic procedures, history of antimicrobial exposure, readmission and relapse of CDAD in both groups were Eur J Clin Microbiol Infect Dis (2009) 28:543–545 DOI 10.1007/s10096-008-0655-7

Severe adenovirus community-acquired pneumonia mimicking Legionella

Dear Editor, I read with interest the report of Drs. Hakim and Tleyjah on severe adenovirus pneumonia in your journal [1]. Their literature review reminds us of the reemergence of adenoviral community-acquired pneumonia (CAP) and that it may mimic other causes of CAP, particularly the atypical pneumonias, e.g., Legionnaire’s disease [2, 3]. As the authors correctly state, adenovirus is the only viral cause of CAP that may resemble bacterial pneumonia with focal/ lobar infiltrates or consolidation. However, as with other viral causes of pneumonia, early the chest X-ray in adenoviral CAP may be normal, even when the patient is short of breath and hypoxemic. The two most likely radiologic presentations of adenovirus is a completely normal chest X-ray with hypoxemia and symptoms of pneumonia or with lobar infiltrate/consolidation. Unlike influenza, routinely associated with leukopenia, adenoviral CAP is associated with a normal or elevated white blood cell (WBC) count, excluding viral influenza. An important clue noted by the authors, which has been our experience as well is the presence of conjunctival suffusion when present. Another important point is that viral pneumonias are ordinarily not accompanied by loose stools/diarrhea [4]. Adenovirus is most likely to be confused in this respect with Mycoplasma pneumoniae or Legionnaire’s disease, which is a common feature of both. Adenoviral CAP in patients with lobar infiltrates mimic Legionnaire’s disease, which may be differentiated from Legionella by the absence of hyponatremia, hypophosphatemia, and highly elevated serum levels [5]. As with conjunctival suffusion, an important clue to adenoviral pneumonia is wheezing, more common in children that, if present, should suggest adenovirus [1]. Recently, we have had a case of adenoviral CAP in a normal host different to that reported by the authors. The patient was a 36-year-old immunocompetent male who presented with severe CAP with a prominent dry cough and headache. On physical examination, he had a questionable faint truncal rash and conjunctival suffusion. Auscultation of the chest revealed no rales. His WBC count was 7.3 K/ mm with monocytosis. Relative lymphopenia was not present. His platelet count was 280,000 K/mm. Because of his headache and possible rash, the question of aseptic meningitis was raised and a lumbar puncture was performed. His cerebrospinal fluid (CSF) showed 8 WBCs/hpf with a normal glucose, protein, and lactic acid level. Head magnetic resonance imaging (MRI) was negative. He was hypoxemic on room air. Both his erythrocyte sedimentation rate (ESR) and liver function tests (LFTs) were unelevated and cold agglutinin titers were negative. His creatine phosphokinase (CPK) was unelevated. Titers for Legionella, M. pneumoniae, and Chlamydia pneumoniae were negative. His Legionella urinary antigen test was negative. His adenovirus titer was elevated 1:128 (normal<1:8). As the authors suggest, adenoviral CAP is probably more common than recognized. It is an easily missed diagnosis and should be included in the differential diagnosis of severe CAP in patients. Early on, in immunocompetent CAP patients without pulmonary infiltrates, the differential diagnosis should include influenza and cytoEur J Clin Microbiol Infect Dis (2009) 28:313–315 DOI 10.1007/s10096-008-0611-6

Amebiasis

Amebiasis is defined as infection with Entamoeba histolytica, regardless of associated symptomatology. In resource-rich nations, this parasitic protozoan is seen primarily in travelers to and emigrants from endemic areas. Infections range from asymptomatic colonization to amebic colitis and life-threatening abscesses. Importantly, disease may occur months to years after exposure. Although E histolytica was previously thought to infect 10% of the world's population, 2 morphologically identical but genetically distinct and apparently nonpathogenic Entamoeba species are now recognized as causing most asymptomatic cases. To avoid unnecessary and possibly harmful therapies, clinicians should follow the diagnostic and treatment guidelines of the World Health Organization.

Application of Isothermal Helicase-Dependent Amplification with a Disposable Detection Device in a Simple Sensitive Stool Test for Toxigenic Clostridium difficile

Enzyme immunoassays (EIAs) are commonly used for the diagnosis of cases of Clostridium difficile-associated diarrhea (CDAD). However, these EIAs have high false-negative rates, even in patients with severe clinical disease. We have developed an IsoAmp CDAD test using a simple and user-friendly procedure to identify toxigenic C. difficile in feces. After DNA extraction from fecal samples, both the conserved sequence of the 5'-end fragment of the C. difficile tcdA toxin gene and competitive amplification internal control sequence were amplified using helicase-dependent amplification. Amplification products were detected using a novel amplicon-containment detection device. The analytical sensitivity of the assay was 20 copies of C. difficile genomic DNA per reaction. Evaluation of the clinical sensitivity and specificity of the IsoAmp CDAD test versus an EIA method using a PCR method as the reference standard revealed 100% sensitivity and 100% specificity for the IsoAmp CDAD test compared with 90.9% sensitivity and 100% specificity for the EIA method. Because the IsoAmp CDAD test requires no expensive equipments for nucleic acid amplification or detection and can be performed on a random access basis, the test provides a practical alternative to immunoassays for the diagnosis of CDAD with improved sensitivity.

Detection of Pseudomonas aeruginosa producing metallo-β-lactamase VIM-2 in a central hospital from Portugal

Pseudomonas aeruginosa remains one of the most important pathogens in the nosocomial setting [1]. P. aeruginosa exhibits intrinsic resistance to several antimicrobial agents. The antipseudomonal β-lactams represent a major weapon against Pseudomonas infections, either for monotherapy or for combination therapy, for which β-lactams almost invariably represent one of the components. Therefore, resistance to these agents constitutes a major challenge for anti-Pseudomonas chemotherapy. Several mechanisms can contribute to β-lactam resistance in P. aeruginosa, including β-lactamase production, outer membrane impermeability and active efflux mediated by RND-type efflux systems [1]. During the last decade, the metallo-β-lactamases (MBLs) have emerged as new threatening mechanisms of broad-spectrum β-lactam resistance in P. aeruginosa. In fact, these enzymes can efficiently degrade virtually all antipseudomonal β-lactams (except aztreonam), while they are not susceptible to therapeutic β-lactamases inhibitors [2]. Based on amino acid sequence homology, these MBLs have been classified into four major types: IMP, VIM, SPM and GIM. Clinical isolates harbouring the MBLs IMP and VIM have been increasingly reported worldwide, mostly in European and Asian countries [2]. This increase in occurrence, types and rate of dissemination of MBLs makes early detection very critical. The benefits of such treatment include the timely implementation of strict infection control practices, as well as clinical guidance. The aim of this study was to determine the presence of these enzymes in imipenem-resistant P. aeruginosa isolates collected at the Centro Hospitalar of Coimbra (CHC), during a two-year period (April 2003 to April 2005) and to ascertain their clonal relationship. CHC is a cluster formed by a central hospital and several specialised units located at distances of less than 8 km, namely, Hospital dos Covoes (central hospital), Instituto Maternal (maternity hospital) and Hospital Pediatrico (paediatric hospital). The Microbiology Laboratory also analysed samples from another hospital (Hospital de Pombal) located 30 km away. Bacterial identification and minimal inhibitory concentrations (MIC) were performed with the MicroScan WalkAway (Dadebehring) system according to the instructions of the manufacturer and API32GN or API PSE systems (bioMerieux). All intermediate strains were considered as nonsusceptible strains. The MICs of β-lactams were also determined by E-test in isolates that presented the enzyme VIM-2. The results were interpreted on the basis of CLSI recommended breakpoints [3]. The screening of MBLs was done by the double combined disk test [4]. Polymerase chain reaction (PCR) analyses for the detection of MBLs genes (blaIMP, blaVIM, blaGIM and blaSPM-1) were carried out for all strains in which the screening test gave positive results [5]. The amplicons were directly sequenced on both DNA strands on an ABI PRISM 377 automated sequencer. The nucleotide and deduced amino acid sequences were analysed with software available online (http://blast.ncbi.nlm.nih.gov/ Eur J Clin Microbiol Infect Dis (2008) 27:1269–1271 DOI 10.1007/s10096-008-0579-2

Audit of antibiotic prescribing in two governmental teaching hospitals in Indonesia

This article estimates the magnitude and quality of antibiotic prescribing in Indonesian hospitals and aims to identify demographic, socio-economic, disease-related and healthcare-related determinants of use. An audit on antibiotic use of patients hospitalized for 5 days or more was conducted in two teaching hospitals (A and B) in Java. Data were collected by review of records on the day of discharge. The method was validated through concurrent data collection in Hospital A. Multivariate logistic regression analysis was performed to determine variables to explain antibiotic prescribing. Prescriptions were assessed by three reviewers using standardized criteria. A high proportion (84%) of 999 patients (499 in Hospital A and 500 in Hospital B) received an antibiotic. Prescriptions could be categorized as therapeutic (53%) or prophylactic (15%), but for 32% the indication was unclear. Aminopenicillins accounted for 54%, and cephalosporins (mostly third generation) for 17%. The average level of antibiotic use amounted to 39 DDD/100 patient-days. Validation revealed that 30% of the volume could be underestimated due to incompleteness of the records. Predictors of antibiotic use were diagnosis of infection, stay in surgical or paediatric departments, low-cost nursing care, and urban residence. Only 21% of prescriptions were considered to be definitely appropriate; 15% were inappropriate regarding choice, dosage or duration, and 42% of prescriptions, many for surgical prophylaxis and fever without diagnosis of infection, were deemed to be unnecessary. Agreement among assessors was low (kappa coefficients 0.13-0.14). Despite methodological limitations, recommendations could be made to address the need for improving diagnosis, treatment and drug delivery processes in this setting.

Detection of multiple respiratory viruses by real-time polymerase chain reaction in infants attending an outpatient clinic

The detection of respiratory viruses in many prospective clinical studies has relied upon detection with standard laboratory assays, such as antigen detection, viral culture, or serology. Data describing the relative importance of community-acquired respiratory viruses, such as rhinovirus, human metapneumovirus (HMPV) [1], and newly described human coronavirus (HCV) subtypes [2], which are not detectable by these standard assays, in healthy young children who are not admitted to the hospital is limited. We developed a novel respiratory real-time reverse transcription (RT) polymerase chain reaction (PCR) panel to detect the presence of HMPV, respiratory syncytial virus (RSV), influenza viruses A and B, parainfluenza virus (PIV) 1, 2, and 3, adenovirus, human coronaviruses Group 1 (229E and NL63) and Group 2 (OC43 and HKU1), and rhinovirus. We conducted a one-year prospective outpatient community-based study of respiratory tract infections (RTIs) in a healthy infant cohort. Our goals were to determine the proportion of viral RTIs caused by these 13 viruses in symptomatic but otherwise healthy infants, as well as to characterize the clinical features of the identified viral infections and quantify the decline in viral load of patients infected with HMPV in this outpatient population. This cohort surveillance study in a population of healthy infants followed for 13 months was conducted between 1 April 2004 and 1 May 2005. Infants between the ages of 0– 6 months were enrolled from the Well Child Clinic at the Madigan Army Medical Center (MAMC) in Tacoma, WA. All subjects were evaluated at each RTI visit by one of three study physicians. Infections were determined to be lower RTIs if bronchiolitis or pneumonia were diagnosed clinically or radiographically by a study physician. We also assessed the presence of a secondary infection requiring antibiotics. During the 13 months of the study, parents called for an appointment in our study clinic if their infant had any respiratory tract symptoms. The clinical data obtained at the study visits for RTIs and by phone follow-up included the duration and complications of the illness, and missed days of work or daycare. Those infants with new onset of at least 2 of 5 respiratory tract symptoms (cough, rhinorrhea, wheezing, fever, and nasal congestion) had a physical Eur J Clin Microbiol Infect Dis (2008) 27:1245–1248 DOI 10.1007/s10096-008-0558-7

Antimicrobial Susceptibility Profile of Pseudomonas aeruginosa Isolates in Egypt

Pseudomonas aeruginosa is a leading cause of nosocomial respiratory tract, urinary tract and skin infections. Data are sparse on the antimicrobial resistance of P. aeruginosa in Egypt. We sought to detect and compare the antimicrobial susceptibility of P. aeruginosa isolates from respiratory tract, urinary tract and skin infections at 3 Egyptian hospitals. Minimum inhibitory concentrations of antibiotics were determined by the agar dilution method. P. aeruginosa respiratory tract infections isolates were 100% resistant to ampicillin, ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate and chloramphenicol, highly resistant to cefuroxime (89%), tetracycline (89%) and azithromycin (84%), and susceptible to norfloxacin (89%), amikacin (84%) and meropenem (68%). P. aeruginosa urinary tract infection isolates were 100% resistant to ampicillin, amoxicillin, chloramphenicol, cefuroxime and tetracycline, highly resistant to amoxicillin/clavulanate (95%), azithromycin (95%), cefalexin (91%) and ampicillin/sulbactam (82%), and susceptible to amikacin (82%), meropenem (73%) and norfloxacin (64%). P. aeruginosa skin infection isolates were 100% resistant to ampicillin and amoxicillin, highly resistant to tetracycline (95%), amoxicillin/clavulanate (95%), cefalexin (87%) and azithromycin (84%), and susceptible to amikacin (87%), norfloxacin (71%) and meropenem (68%). The anti-pseudomonal effect of antibiotics varied among different infection sites only for ampicillin/sulbactam, cefoperazone or chloramphenicol but not with the other tested antibiotics. Norfloxacin and amikacin could be used for initial therapy for P. aeruginosa mediated respiratory tract infections. Amikacin, meropenem and norfloxacin could be used for P. aeruginosa mediated urinary tract and skin infections. Such studies are essential to determine the current guidelines for empirical therapy regimens, which vary by location, and help with the establishment of effective infection control measures.

Activity of serum concentrations of daptomycin vs. vancomycin against vancomycin-susceptible and resistant Enterococcus faecium in the presence of albumin physiological concentrations: an in vitro pharmacodynamic simulation

Killing curves with daptomycin (56 μg/ml, the Cmax after 4 mg/kg) in media containing albumin physiological concentrations showed bactericidal activity against Enterococcus faecium, but due to its high protein binding (91.7%), cidal activity was delayed when compared to that in media without albumin [1]. This study explores the activity over 24 h of simulated serum concentrations of daptomycin (after 6 mg/kg od regimen) vs. vancomycin (1,000 mg bid regimen) against two vancomycin-susceptible but tolerant and one resistant E. faecium in an in vitro pharmacodynamic device using media containing albumin physiological concentrations. A one-compartment dynamic model was used to expose bacteria to changing study drug concentrations [2] using Mueller Hinton broth supplemented with albumin (4 g/dl). In daptomycin simulations, media was calcium-adjusted by adding 100 μg/ml of Ca [3]. Albumin concentrations were determined by the bromcresol green method, ionized calcium concentrations were measured using the AU2700 system (Olympus Diagnostic, Barcelona, Spain), and the total Ca by the Arsenazo III method (BioSystems, Barcelona, Spain). The distribution volume of vancomycin (66.5 ml) and daptomycin (100 ml) were calculated using the expression Vd=Cl/Ke to simulate the half-lives of vancomycin [4] and daptomycin [5]. Pharmacokinetic profiles in serum after 1,000 mg vancomycin [4] bid and 6 mg/kg daptomycin [5] od were simulated over 24 h. The target pharmacokinetic parameters [4, 5] were: Cmax=43.05 μg/ml, Cmin=10.8 μg/ml, and t1/2=6 h for vancomycin; and Cmax=86.4 μg/ml, Cmin=10.7 μg/ml, and t1/2=8 h for daptomycin. The initial inoculum was 8.9×10–1.8×10 cfu/ml. Samples (0.5 ml) were collected at 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, serially diluted in 0.9% sodium chloride, washed in normal saline, vacuum-filtered through 0.45-micron membranes to avoid the carry-over effect, and the filters plated for colony counting (detection limit=50 cfu/ml). Experiments were performed in triplicate. Differences between initial inocula and colony counts at the different timepoints (expressed as % reduction) and the times to obtain 90% (T90%), 99% (T99%), and 99.9% (T99.9%) reductions were determined. Aliquots (0.5 ml) were taken at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h for the measurement of concentrations by bioassay [2] (detection limit: 0.5μg/ml daptomycin, 4μg/ml vancomycin). The total and extrapolated free-drug concentrations (adjusting by 91.7% protein binding of daptomycin and 36.9% of vancomycin) were analyzed by a non-compartmental approach using the WinNonlin Professional program (Pharsight, Mountain View, CA). The area under the concentration–time curve (AUCall) and AUC0–24h/MIC for the total and free drugs were calculated. Changes in viable colony counts at 12 h and 24 h were compared by the two-tailed t-test. The experimental pharmacokinetic parameters were: Cmax=44.5±2.6 μg/ml, Cmin=10.6±1.0 μg/ml, t1/2=6.0±0.5 h and AUCall=286.5±13.1 μg/ml×h for vancomycin; and Cmax=85.7±5.8 μg/ml, Cmin=9.4±1.1 μg/ml, t1/2=8.1±0.2 h and AUCall=852.7±15.8 μg/ml×h for daptomycin. The measured albumin concentrations were 3.9±0.21 g/dl, meaEur J Clin Microbiol Infect Dis (2008) 27:1009–1011 DOI 10.1007/s10096-008-0525-3

OXYSTEROLS INFLUENCE HEPATIC CELLS HOMEOSTASIS

gastric mucosa due to H. pylori infection may result in decreased numbers of ghrelin producing cells and lower plasma ghrelin levels [7,9]. In contrast, there was not significant difference in plasma ghrelin levels between H. pylori infected patients and H. Pylori uninfected patients in our study; 32,56±21,97 and 34,04±20,59 respectively (p:0,75), similar with another Turkish study [8]. In conclusion, according to our results, there is not a relation between plasma ghrelin levels and H. pylori infection. These conflicting results may be related to different H. pylori strains, as in Turkey the prevalence of the strains, such as CagA, that cause atrophic gastritis is lower [11]. Moreover; differences in studied populations in terms of race, sample size, nutritional status and habits can also affect results. Decreasing plasma ghrelin levels in H. pylori infected subjects can be compensated by other extra gastric ghrelin producing tissues, convenient to our results. All together, further studies are needed to demonstrate the association between H. pylori infection and plasma ghrelin levels.

Oral therapy for urinary tract infections in infants aged 3 to 12 months

Urinary tract infections (UTI) are one of the most common serious bacterial infections in infants. Guidelines in the past recommended intravenous (IV) antibiotics for the treatment of febrile UTI [1, 2]. However, a randomized controlled trial and a Cochrane systematic review demonstrated that oral cefixime is a safe and effective treatment [3, 4]. Oral amoxicillin-clavulanic acid has also been demonstrated to be as effective as initial IV antibiotics [5]. Oral therapy would preclude the costs associated with hospitalization. The choice of initial antimicrobial should be guided by local resistance patterns [5]. In our environment, cefixime or amoxicillin-clavulanic acid could be regarded as safe options [6, 7]. The aim of our study was to evaluate response to oral amoxicillin-clavulanic acid in children aged 3 to 12 months with their first episode of UTI and to compare the response with that of a historical cohort of patients treated in our center with IV therapy. We conducted a prospective study between November 2005 and September 2006. Children aged 3 to 12 months were considered to be eligible if they had an axillary temperature of 38°C or more at presentation or within 24 h and were suspected of having a UTI because of the presence of any degree of bacteriuria. Final eligibility required that there be a positive urine culture (≥10,000 colony-forming units/ml, single pathogen) from a specimen obtained by catheter. Children were excluded if they had hypersensitivity to penicillin, a history of UTI or abnormalities of the urinary tract, had received a systemic antimicrobial in the previous 48 h, had an underlying chronic disease, or had not completed the follow-up protocol. Infants assessed as toxic, dehydrated, or unable to retain oral intake were also excluded from the study. All children included in the study received oral amoxicillinclavulanic acid (Augmentine® oral solution 100/12.5, GlaxoSmithKline, Madrid, Spain) 40 mg/kg/day for 10 days. We modified the treatment in children with persistent fever (>38°C) in the 72 h after the start of treatment and those with intolerance or an adverse reaction to the drugs used. Renal ultrasound and a voiding cystourethrogram (VCUG) were performed within 10 days from the diagnosis and a Tcdimercaptosuccinic acid (Tc-DMSA) renal scan was performed 6 months later. We also conducted a retrospective study including infants with UTI aged 3 to 12 months treated with IV amoxicillin-clavulanic acid 100 mg/kg/day for 3 days or until they became afebrile, before the implementation of the new guidelines, from June 2004 to November 2005. The inclusion and exclusion criteria were the same as for the prospective study group. Outcomes of the group treated with oral amoxicillin-clavulanic acid were comparedwith outcomes of the retrospective group. Eur J Clin Microbiol Infect Dis (2008) 27:887–889 DOI 10.1007/s10096-008-0517-3

Human α-Defensins Inhibit Clostridium difficile Toxin B

Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Inactivation of C difficile toxins by alpha-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine K(m) and median inhibitory concentration (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K(m) values for uridine 5'-diphosphate-glucose up to 10-fold. The IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.

Implementation of an infection control programme to limit the spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in a Belgian university hospital

Extended-spectrum β-lactamase-producing Enterobacteriaceae (E-ESBLs) are an increasing problem all over the world due to their importance in nosocomial infections and these bacteria have been the cause of several important outbreaks. 1 Pena C. Pujol M. Ardanuy C. et al. Epidemiology and successful control of a large outbreak due to K. pneumoniae producing extended-spectrum β-lactamases. Antimicrob Agents Chemother. 1998; 42: 53-58 PubMed Google Scholar , 2 Moriguchi N. Itahashi Y. Tabata N. et al. Outbreak of CTX-M-3-type extended-spectrum beta-lactamase-producing Enterobacter cloacae in a pediatric ward. J Infect Chemother. 2007; 13: 263-266 Abstract Full Text PDF PubMed Scopus (14) Google Scholar , 3 Manzur A. Tubau F. Pujol M. et al. Nosocomial outbreak due to extended-spectrum-beta-lactamase-producing Enterobacter cloacae in a cardiothoracic intensive care unit. J Clin Microbiol. 2007; 45: 2365-2369 Crossref PubMed Scopus (47) Google Scholar In Belgium, nosocomial infections with E-ESBLs have gradually increased over the last 15 years. 4 De Gheldre Y. Glupczynski Y. Struelens M. De Mol P. Emergence of E.aerogenes as a major antibiotic resistant nosocomial pathogen in Belgian hospitals. Clin Microbiol Infect. 1999; 5: 622-627 Crossref PubMed Scopus (25) Google Scholar

Mannitol and inflammatory markers in the cerebral spinal fluid of HIV-infected patients with cryptococcal meningitis

Mannitol is a polyol produced by Cryptococcus neoformans and other fungi, including Aspergillus and Saccharomyces species [1]. A by-product of fungal metabolism, mannitol acts as a scavenger for extracellular free radicals protecting the organism from the environment and assists in the evasion of host phagocytic destruction [2–4]. Mannitol has been detected at higher concentrations in the cerebrospinal fluid (CSF) of animals with cryptococcal meningitis (CM) than in uninfected controls [4]. The accumulation of mannitol in the CSF may exert a significant osmotic effect in the central nervous system of patients with C. neoformans meningitis, due to its inability to cross the blood–brain barrier and its resistance to host metabolism [5]. The concentration of mannitol in the CSF has been correlated with both cryptococcal antigen titre and with quantitative cryptococcal culture in animal models of CM [4]. Megson et al. [5] were able to detect mannitol in 19 of 20 CSF samples from HIV-infected patients with CM; however, no comparisons were made to the levels in uninfected controls or to disease severity. CSF was collected with informed consent from HIVinfected patients for whom the diagnosis of CM was being considered. Mannitol may play a role in the development of cerebral oedema and increased intracranial pressure (ICP) seen in patients with CM, and its presence during infection may impact the host local inflammatory response. We hypothesised that an elevated ICP, as measured by the opening pressure (OP), and local central nervous system (CNS) inflammatory parameters from confirmed cases of CM would correlate to the CSF concentration of mannitol and that these levels would be higher than in HIV-infected patients without CM. CM diagnosis was made based on the clinical signs and symptoms of meningitis and either confirmed growth of C. neoformans from CSF or blood and/or a detectable CSF cryptococcal antigen titre of 1:2 or higher. Control patients presented with a clinical suspicion for meningitis, but their CSF and blood cultures did not yield C. neoformans and their CSF cryptococcal antigen tests were negative. Patients with a prior history of CM were excluded. CSF samples were stored at −80°C until batched analysis could be performed. The concentration (μg/ml) of mannitol was determined by the gas–liquid chromatography method previously described by Wong et al. [4]. Undiluted CSF samples were assayed in duplicate for selected inflammatory parameters Eur J Clin Microbiol Infect Dis (2008) 27:477–479 DOI 10.1007/s10096-008-0462-1

The presence of infectious extracellular Francisella tularensis subsp. novicida in murine plasma after pulmonary challenge

Francisella tularensis is a gram-negative, facultative intracellular bacterium and is the causative agent of the zoonotic disease tularemia [1]. F. tularensis has been considered a potential biological weapon due to its low infectious dose and high mortality rate [2]. F. tularensis can be classified into several subspecies, including those relevant to human disease: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B); F. novicida and F. mediasiatica [3]. However, most of our knowledge about the pathogenesis of Francisella and the immune responses to the infection have come from studies of F. tularensis LVS (derived from holartica) and F. novicida [4–6]. Both organisms are attenuated in humans, while retaining virulence in mice. It is well established that Francisella enters and replicates within host cells, and a strong research focus has been on deciphering the mechanisms for intramacrophage growth. However, little is known about the extracellular phase of Francisella. F. tularensis has been cultured from blood of infected animals and humans, although the reported cases have been rare [7, 8]. Specifically, Long et al. [8] demonstrated that LVS could be recovered from plasma prepared from mice 72 h after intravenous infection. These extracellular bacteria accounted for less than 1% of total LVS within the blood. However, a recent study by Forestal et al. [9] indicated a much higher percentage (>50%) of extracelluar Francisella (type A SCHU S4 and LVS) within plasma of mice infected intradermally or intranasally. Here, independently, we confirm the presence of extracellular Francisella in plasma after intranasal F. novicida challenge in a murine model of pulmonary tularemia [6]. Moreover, we demonstrate that the extracellular Francisella in plasma is highly infectious and that this phase may contribute to the rapid dissemination of the bacterium from lungs to the liver after pulmonary infection. F. novicida strain U112 shares a high degree of antigenic and genetic similarity with F. tularensis type A and B [10] and is highly virulent in mice with a LD50 of approximately 10 colony forming units (CFU) by the intranasal (i.n.) route [6]. In this study, we monitored the replication and dissemination of F. novicida in mice. Bacteria were grown at 37°C in Trypticase Soy Broth (TSB) supplemented with 0.1% cysteine. The concentrations of the bacterial inocula were determined by serial dilution plating on Trypticase Soy Agar (TSA). Female BALB/c mice (6–8 weeks old, obtained from the Jackson Laboratory, Bar Harbor, ME) were challenged i.n. with 400 CFU (40 LD50). All animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines of the University of Texas at San Antonio. Bacterial loads in the lungs and livers were assessed by plating tissue homogenates on TSA. Within 24 h of pulmonary challenge, a large number of F. novicida, approximately 1×10 CFU/g tissue, was observed in the lungs, which increased to 1×10 CFU/g tissue by 48 h and then decreased to 1×10 CFU/g tissue by 72 h. In the livers of infected mice, there was a progressive increase in bacterial load with levels rising from 100 CFU/g tissue at 24 h to approximately 1×10 CFU/g tissue by 72 h (Fig. 1a). These results demonstrate dissemination of F. novicida from the lungs to the secondary organs after pulmonary exposure. To determine a probable mechanism for the rapid spread to the secondary organs, blood was collected from the orbital plexus at 24, 48 and 72 h Eur J Clin Microbiol Infect Dis (2008) 27:323–325 DOI 10.1007/s10096-007-0434-x