Intestinal colonization with resistant bacteria: a prognostic marker of mortality in decompensated cirrhosis.

Infections due to drug-resistant (DR) bacteria are increasingly recognized as an emerging problem worldwide. Asymptomatically colonized patients may contribute to the reservoir in the hospital setting, causing both horizontal transmission and endogenous infections. We aimed to evaluate the prevalence of intestinal colonization with DR bacteria on subsequent clinical infection development and prognosis in patients with decompensated cirrhosis. One hundred seven patients without infection at baseline were screened and prospectively followed-up for 3 months. Among the patients screened, DR bacteria were isolated in 47 (43.9%), 14 colonized with multidrug- (MDR) and 33 with extensively drug (XDR)-resistant bacteria or a mixture of MDR/XDR bacteria. Severity of liver disease and demographic characteristics were similar among groups. The 20 (42.6%) with DR vs 14 (23.3%) without had hepatic encephalopathy and/or spontaneous bacterial peritonitis episodes over the past 6 months (p = 0.034). One third of both DR and non-DR groups developed infection during follow-up but in only 7 and 5, respectively, the infection was microbiologically documented. In a 3-month-follow-up period, mortality was higher in patients colonized with XDR compared to those without (log rank p = 0.027). In multivariate analysis, colonization with XDR bacteria [HR = 1.074, (CI:1.024-1.126), p = 0.003] and MELD score [HR = 2.579 (1.109-5.996), p = 0.028] were independently associated with low survival. Asymptomatic GI colonization with DR bacteria is a risk factor for increased mortality in decompensated cirrhosis. Frequent hospitalizations for complications of the underlying disease and selective pressure induced by the use of antimicrobials are probably the main determinants.

Infection control interventions affected by resource shortages: impact on the incidence of bacteremias caused by carbapenem-resistant pathogens.

We evaluated an infection control (IC) program influenced by personnel and material resource shortages on the incidence of bloodstream infections (BSI) due to carbapenem-resistant Klebsiella pneumoniae (CRKP), Acinetobacter baumannii (CRAB), and Pseudomonas aeruginosa (CRPA) in an endemic region. Between January 2010 and December 2015, all BSI episodes caused by CRKP, CRAB, and CRPA were recorded. An IC bundle was implemented in January 2012. We evaluated the effect of the interventions on BSI rates between the pre-intervention (2010-2011) and intervention (2012-2013) periods, using an interrupted time-series model. From 2014, when interventions were still applied, BSI incidence was gradually increased. For this reason, we evaluated with a linear mixed effects model several factors possibly contributing to this increase for the years 2012-2015, which was considered as the intervention/follow-up period. During the study period, 351 patients with BSI were recorded, with a total of 538 episodes; the majority (83.6%) occurred in the intensive care unit (ICU). The BSI incidence rate per year during 2010-2015 for ICU patients was 21.03/19.63/17.32/14.45/22.85/25.02 per 1000 patient-days, respectively, with the reduction in BSI levels after the start of intervention marginal (p = 0.054). During the follow-up period (2014-2015), the most influential factors for the increased BSI incidence were the reduced participation in educational courses and compliance with hand hygiene. The implementation of IC interventions reduced the BSI incidence rates, particularly for ICU patients. However, factors possibly related to the restrictions of human and material resources apparently contributed to the observed expansion of BSI in our endemic setting.

First report of Dientamoeba fragilis infection explaining acute non-specific abdominal pain.

Dientamoeba fragilis is now considered a potentially emerging gastrointestinal pathogen in both developing and developed countries. We first report an autochthonous case of D. fragilis infection in Greece. A 49-year-old female with acute non-specific abdominal pain required emergency surgical admission for active observation and repeated assessment. To the best of our knowledge, this is the first reported case of acute unexplained abdominal pain finally attributed to D. fragilis infection using microscopic and molecular methods.

Spread of a carbapenem- and colistin-resistant Acinetobacter baumannii ST2 clonal strain causing outbreaks in two Sicilian hospitals.

BACKGROUND: Infections caused by multidrug-resistant (MDR) Acinetobacter baumannii have become an important healthcare-associated problem, particularly in intensive care units (ICUs). AIM: To investigate the emergence of carbapenem- and colistin-resistant A. baumannii infections in two Sicilian hospitals. METHODS: From October 2008 to May 2011, a period which included two Italian Nosocomial Infections Surveillance in ICUs network (SPIN-UTI) project surveys, all carbapenem-resistant A. baumannii isolates from the ICUs of two hospitals in Catania, Italy, were prospectively collected. Minimum inhibitory concentrations (MICs) were measured by agar dilution, and phenotypic testing for metallo-ß-lactamase (MBL) production was performed. Carbapenem resistance genes and their genetic elements were identified by polymerase chain reaction and sequencing. Genotypic relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. Patient-based surveillance was conducted using the SPIN-UTI protocol and previous antibiotic consumption was recorded. FINDINGS: Twenty-six carbapenem-resistant A. baumannii were identified. Imipenem and meropenem MICs ranged from 4 to >32 mg/L, and 15 isolates exhibited high-level colistin resistance (MICs >32 mg/L). PFGE demonstrated that all isolates belonged to a unique clonal type and were assigned to ST2 of the international clone II. They harboured an intrinsic blaOxA-51-like carbapenemase gene, blaOxA-82, which was flanked upstream by ISAba1. CONCLUSIONS: The dissemination of clonally related isolates of carbapenem-resistant A. baumannii in two hospitals is described. Simultaneous resistance to colistin in more than half of the isolates is a problem for effective antibiotic treatment. Prior carbapenem and colistin consumption may have acted as triggering factors.

Ongoing outbreak of West Nile virus infection in humans, Greece, July to August 2011.

Between 16 July and 21 August 2011, 31 cases of West Nile neuroinvasive disease were reported from four regions in Greece. Of these, 17 occurred in districts that had not been affected in 2010. The reoccurrence of human cases in two consecutive years (following the large 2010 outbreak) and the spread of the virus in new areas suggest that West Nile virus is established in Greece, and its transmission may continue to occur in the future.

The effect of homocysteine on the clinical outcomes of ventilated patients with severe sepsis.

BACKGROUND: There is considerable evidence that elevated plasma homocysteine levels are associated with a prothrombotic milieu, whereas activation of the coagulation cascade is an important component of the pathogenesis of sepsis. The protein C pathway has been reported to play a central role both in the propagation of sepsis and a hyperhomocysteinemia-induced hypercoagulable state. Our primary aim was to measure plasma homocysteine levels in mechanically ventilated patients with severe sepsis/septic shock and to assess the association of these levels with relevant clinical outcomes. METHODS: The study cohort included 102 mechanically ventilated patients with severe sepsis or septic shock. Demographics, comorbidities, clinical data and severity scores were recorded. Plasma homocysteine, vitamin B12, folate, creatinine, and protein C levels were measured in all study subjects upon enrollment, and genotyping for the C677T and A1298C polymorphisisms of the methylenetetrahydrofolate reductase (MTHFR) gene and for factor V Leiden (FVL) mutations was performed as well. The primary outcomes were mortality at 28 and 90 days; secondary outcomes included the number of days without renal or cardiovascular failure and the ventilator-free days during the study period. RESULTS: Homocysteine levels were not significantly associated with any primary or secondary outcomes in the multivariable analysis. In addition, a synergistic effect of homocysteine with protein C levels was not detected. CONCLUSION: Our data suggest that plasma homocysteine levels may not inform the prognosis of mechanically ventilated patients with severe sepsis/septic shock.

Viral agents of acute gastroenteritis in hospitalized children in Greece.

A 6-year study of stool samples from 4604 children hospitalized for acute gastroenteritis was conducted to investigate the role of enteric viruses as a cause of gastroenteritis in north-west Greece. Rotaviruses, noroviruses, adenoviruses and astroviruses were detected in 21.35%, 4%, 3.5% and 2.35%, respectively, by enzyme immunoassays and molecular techniques. Molecular techniques enhanced overall diagnostic efficacy by 2.5%, and by c. 10% each for rotavirus and adenovirus. Rotavirus was the leading cause of viral gastroenteritis, usually associated with severe illness. Mixed infections were found in 4.4% of positive specimens, and rotavirus plus astrovirus represented the most frequent co-infection (55.5%). This first study on the epidemiology of viral gastroenteritis in Greece shows that recent advances in the diagnosis of viral enteropathogens may have only marginal effects on overall diagnostic efficacy, and thus the impact of viral agents causing sporadic gastroenteritis in public health cannot be fully evaluated.

Chlamydia trachomatis infections in Greece: first prevalence study using nucleic acid amplification tests.

The present retrospective study was initiated to determine the prevalence of Chlamydia trachomatis and to assess the risk factors for infection in adult women and men presenting to general practitioners, gynecologists, dermatologists, and family-planning centers in Greece. The study was carried out in four different Greek hospital centers using highly sensitive nucleic acid amplification techniques. Altogether, 16,834 women and 1,035 men were enrolled from October 1998 to April 2004. Two types of specimens were collected from each patient: cervical swabs from women, urethral swabs from men, and first-catch urine from women and men. All specimens were examined with the Cobas Amplicor C. trachomatis polymerase chain reaction assay (Roche Molecular Systems, Branchburg, NJ, USA) or the LC x C. trachomatis ligase chain reaction assay (Abbott Laboratories, Abbott Park, IL, USA). Demographic and behavioral data were collected by clinicians using a standardized questionnaire. A total of 704 (3.9%) patients were infected with C. trachomatis. The prevalence among female patients was 3.5% and that among male patients 11.2%. Among infected patients, 88% were under 30 years of age, 71% reported more than one sexual partner, and 91% reported a new sexual partner within the last year. In conclusion, the prevalence of C. trachomatis infection in Greece is low. Young age and new and multiple sexual partners within the last year were factors consistently associated with an increased risk of chlamydial infection.

Evaluation of a commercially available reverse transcription-PCR enzyme immunoassay (Enterovirus Consensus kit) for the diagnosis of enterovirus central nervous system infections.

A commercial reverse transcription (RT)-PCR amplification method was compared with culture for the diagnosis of enterovirus meningitis. In total, 99 cerebrospinal fluid (CSF) specimens were examined with the Enterovirus Consensus kit and shell vial culture. RT-PCR allowed the amplification of enterovirus cDNA and its detection in a microtitre plate by hybridisation. Clinical information and CSF analysis were used to resolve the discrepancy in results. The detection limit of the RT-PCR assay was determined with the Third European Union Concerted Action Enterovirus Proficiency Panel. There were 34 true-positive CSF specimens. Of these, RT-PCR detected 33 (sensitivity 97%), while culture detected 19 (sensitivity 54.5%). RT-PCR failed to detect one culture-positive specimen that contained inhibitors. When samples from the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, the RT-PCR method gave identical results to those expected. The Enterovirus Consensus kit was rapid and statistically more sensitive than culture (p < 0.01) for the detection of enteroviruses in CSF, and may offer considerable benefits in the clinical management of patients with enterovirus meningitis.

Fatal meningitis due to Listeria monocytogenes in elderly patients with underlying malignancy.

Adult patients with malignancies are considered to be at a high risk for Listeria monocytogenes meningitis. The Microbiology Laboratory's database of the University Hospital of Ioannina, Greece, was searched for cases of L. monocytogenes during the period from January 1990 to December 2002. Listerial meningitis occurred in three patients: one with brain tumour, one with chronic lymphocytic leukaemia, and one with non-Hodgkin's lymphoma. All the patients were older than 70 and they were actively receiving therapy for their malignancy. L. monocytogenes type 4b was isolated from blood and cerebrospinal fluid. All were treated with ampicillin and gentamicin, but they died shortly after the initiation of the treatment. Experience with the three present cases indicated the high mortality rate due to listerial meningitis in this immunosuppressed population. So, listeriosis should be suspected in patients with meningitis and underlying malignancy. Since meningitis due to L. monocytogenes is not distinguishable clinically from other types of bacterial meningitis, it is recommended to cover Listeria in the initial empirical therapy of bacterial meningitis in immunosuppressed patients.

Application of a polymerase chain reaction enzyme immunoassay in peripheral whole blood and serum specimens for diagnosis of acute human brucellosis.

A simple polymerase chain reaction-enzyme immunoassay (PCR-EIA) was employed for the rapid laboratory diagnosis of human brucellosis directly from peripheral blood. Whole blood and serum specimens were collected from 243 patients with acute brucellosis as determined by blood culture, serological tests, and the patients' clinical characteristics and from a control group of 50 healthy individuals. Diagnosis of brucellosis was established in 179 cases by isolation of Brucella spp. in blood culture and in 64 cases by clinical signs and serological investigation. Following the amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenic membrane protein specific for the Brucella genus, the amplified product was detected in a microtiter plate by hybridization. Two hundred forty-one of the 243 patients tested had detectable Brucella DNA in either whole blood or serum specimens: 149 (61.3%) patients were positive in both whole blood and serum specimens, 43 (17.7%) were positive in serum specimens only, and 49 (20.2%) were positive in whole blood specimens only. The diagnostic specificity of the PCR-EIA assay for both specimen categories was 100%, while the sensitivity was 81.5% for whole blood specimens, 79% for serum specimens, and 99.2% for whole blood and serum specimens combined. The results suggest that the detection of Brucella DNA in whole blood and serum specimens by PCR-EIA assay is a sensitive and specific method that could assist the rapid and accurate diagnosis of acute human brucellosis.

Four-year experience of use of the Cobas Amplicor system for rapid detection of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens in Greece.

To evaluate the experience of a clinical microbiology laboratory with a DNA amplification assay for routine detection of Mycobacterium tuberculosis, the Cobas Amplicor Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) assay (Roche Diagnostics Systems, USA) was performed on 7,722 respiratory and 1,451 nonrespiratory specimens collected from 3,321 patients. The results were compared with those of culture in conventional Lowenstein-Jensen medium, culture in the MB/BacT system (Organon Teknika, France), and clinical investigations. A total of 240 of the 254 respiratory specimens culture positive for Mycobacterium tuberculosis were also positive in the PCR assay. Of the 7,300 culture-negative specimens, 45 (0.6%) were positive in the PCR. After detailed interpretation, the overall sensitivity, specificity, and positive and negative predictive values of the PCR assay were 84.5, 99.8, 94.1, and 99.4%, respectively, for respiratory specimens. The PCR assay was more sensitive for smear-positive respiratory specimens (97.1%) than for smear-negative respiratory specimens (48.6%). Of the 18 culture-positive (smear-negative) nonrespiratory specimens, 9 were positive in the PCR. None of the 1,384 culture-negative nonrespiratory specimens were positive in the PCR. The inhibition rates detected by the internal control of the test were 2.2% for respiratory specimens and 3.4% for nonrespiratory specimens. After resolving the discrepancies, the overall sensitivity, specificity, and positive and negative predictive values of the PCR assay were 82.5, 99.8, 94.3, and 99.4%, respectively, when compared to the results of diagnostic culture. In conclusion, the use of the Cobas Amplicor MTB-PCR assay might enable clinical microbiology laboratories with considerable previous experience in molecular biology testing to perform PCR and confirm tuberculosis infection immediately, leading to improved patient management.

Molecular typing of Candida isolates from patients hospitalized in an intensive care unit.

OBJECTIVES: The aim of our study was the molecular typing of 40 clinical isolates of Candida spp. obtained from patients with burns or trauma hospitalized in the intensive care unit of a general hospital. METHODS: Isolates were recovered from blood, deep trauma, urine, sputum or from environment within a short period of time (4 months). The yeasts were identified using commercial yeast identification kits as C. albicans (17 isolates), C. tropicalis (16 isolates) and C. parapsilosis (10 isolates). The epidemiological relation of the isolates was tested with the Random Amplified Polymorphic DNA assay using three or four arbitrary chosen primers. RESULTS: All C. albicans isolates presented distinct RAPD profiles, C. tropicalis isolates presented both the same and distinct RAPD patterns and the C. parapsilosis isolates presented the same RAPD pattern. All the environmental isolates were identified as C. parapsilosis and they had the same RAPD pattern as C. parapsilosis clinical isolates. Candida parapsilosis delineation was confirmed with PFGE. CONCLUSIONS: The colonization/infection with C. albicans was endogenous, the C. tropicalis colonization/infection was both endogenous and exogenous, and the C. parapsilosis colonization/infection had an environmental origin.

Human immunodeficiency virus (HIV) protease inhibitors have no effect on hepatitis C virus (HCV) serum levels of HIV-HCV co-infected patients.

Ten severely immunocompromised HIV-HCV co-infected patients were enrolled in a quantifiable HCV-RNA assay. Serum alanine aminotransferase, HCV-RNA levels and HIV viral loads were determined at baseline, at month three and at month six after initiation of a highly active antiretroviral therapy including an HIV protease inhibitor. HCV genotypes were determined using a line probe assay kit. Our results suggested that this therapy did not result in lower HCV viraemia, whatever the HCV genotypes, and probably had no effect on the outcome of chronic viral hepatitis C. As our patients were severely immunocompromised and their mean increase of CD4 cell counts was less than 50/mm(3), we cannot reach any conclusions about the impact of the improvement of immune status on the HCV-RNA load.

Treatment of disseminated Mycobacterium genavense infection in a murine model with ciprofloxacin, amikacin, ethambutol, clarithromycin and rifabutin.

Mycobacterium genavense is a recently described agent which can induce disseminated infections in patients with AIDS. Up to now, no standard approach to treatment has been defined and patients have been treated empirically with antibiotics used for treating infections caused by other nontuberculous mycobacteria. In this study, we compared the effectiveness of ciprofloxacin, amikacin, ethambutol, clarithromycin and rifabutin in the treatment of an animal model of M. genavense infection in C57BL/6 mice. Antimycobacterial treatment was started 4 weeks after an intravenous bacterial challenge and was continued for 30 days. Treated and control mice were killed at days 15 and 30 of treatment and the number of viable bacteria in their spleens was counted. Treatment with clarithromycin (50 mg/kg/day sc) and rifabutin (20 mg/kg/day po) was found to decrease the bacterial counts in the spleens significantly as early as 15 days after the onset of treatment (P < 0.01). The effect of treatment was more pronounced after 30 days of treatment (P < 0.001). Amikacin (25 mg/kg/day sc) and ethambutol (50 mg/kg/day sc) were found to decrease significantly the cfu in the spleens only after 30 days of treatment (P < 0.01). Ciprofloxacin (25 mg/kg/day sc) was ineffective in the experimental conditions used here.

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Greece.

There is a geographic distribution of Mycobacterium tuberculosis strains with various rpoB gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoB mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect rpoB mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct rpoB mutations in codons Ser531, His526, and Asp516 were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser531 and His526 accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosis strains can have subclones bearing distinct mutant rpoB alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosis before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.