Central-line-associated bloodstream infections, multi-drug-resistant bacteraemias and infection control interventions: a 6-year time-series analysis in a tertiary care hospital in Greece.

BACKGROUND: Central-line-associated bloodstream infections (CLABSIs) are serious healthcare-associated infections with substantial morbidity and hospital costs. AIM: To investigate the association between the incidence of CLABSIs, the implementation of specific infection control measures, and the incidence of multi-drug-resistant (MDR) bacteraemias in a tertiary care hospital in Greece from 2013 to 2018. METHODS: Analysis was applied for the following indices, calculated monthly: CLABSI rate; use of hand hygiene disinfectants; isolation rate of patients with MDR bacteria; and incidence of bacteraemias [total Gram-negative carbapenem-resistant Acinetobacter baumanii, carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Klebsiella pneumoniae; and Gram-positive meticillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci]. FINDINGS: The total number of bacteraemias from carbapenem-resistant Gram-negative pathogens was significantly correlated with an increased CLABSI rate for all (total) hospital departments [incidence rate ratio (IRR) 1.17, 95% confidence interval (CI) 1.05-1.31, P=0.006] and the adult intensive care unit (ICU) (IRR 1.37, 95% CI 1.07-1.75, P=0.013). In the adult ICU, every increase in the incidence of each resistant Gram-negative pathogen was significantly correlated with a decreased CLABSI rate (carbapenem-resistant A. baumanii: IRR 0.59, 95% CI 0.39-0.90, P=0.015; carbapenem-resistant K. pneumoniae: IRR 0.48, 95% CI 0.25-0.94, P=0.031; carbapenem-resistant P. aeruginosa: IRR 0.54, 95% CI 0.33-0.89, P=0.015). The use of hand disinfectants was correlated with a decreased CLABSI rate 1-3 months before the application of this intervention for all (total) hospital departments (IRR 0.80, 95% CI 0.69-0.93, P=0.005), and for scrub disinfectants in the current month for the adult ICU (IRR 0.34, 95% CI 0.11-1.03, P=0.057). Isolation of patients with MDR pathogens was not associated with the incidence of CLABSIs. CONCLUSION: Hand hygiene was associated with a significant reduction in the incidence of CLABSIs at the study hospital. Time-series analysis is an important tool to evaluate infection control interventions.

Evaluation of the limiting antigen avidity EIA (LAg) in people who inject drugs in Greece.

This analysis assessed the utility of the limiting antigen avidity assay (LAg). Samples of people who inject drugs (PWID) in Greece with documented duration of HIV-1 infection were tested by LAg. A LAg-normalized optical density (ODn) ⩽1·5 corresponds to a recency window period of 130 days. The proportion true recent (PTR) and proportion false recent (PFR) were estimated in 28 seroconverters and in 366 samples collected >6 months after HIV diagnosis, respectively. The association between LAg ODn and HIV RNA level was evaluated in 232 persons. The PTR was 85·7%. The PFR was 20·8% but fell to 5·9% in samples from treatment-naive individuals with long-standing infection (>1 year), and to 0 in samples with the circulating recombinant form CRF35 AD. A LAg-based algorithm with a PFR of 3·3% estimated a similar incidence trend to that calculated by analyses based on HIV-1 seroconversions. In recently infected persons indicated by LAg, the median log10 HIV RNA level was high (5·30, interquartile range 4·56-5·90). LAg can help identify highly infectious HIV(+) individuals as it accurately identifies recent infections and is correlated with the HIV RNA level. It can also produce reliable estimates of HIV-1 incidence.

Estimating the treatment cascade of chronic hepatitis B and C in Greece using a telephone survey.

Accurate diagnosis and treatment rates for chronic hepatitis B (HBV) and C virus (HCV) infections are usually missing. Aim of this study was to estimate the HBV and HCV treatment cascade (proportion and absolute numbers of tested, aware/unaware, infected and treated) in Greek adults. A telephone survey was conducted in a sample representative of the Greek adult general population. Prevalence rates were age-standardized for the Greek adult population and corrected for high-risk individuals not included in the survey. Of the 9974 participants, 5255 (52.7%) had been tested for HBV and 2062 (20.7%) for HCV with the proportion varying according to age and being higher in middle-age groups (P < 0.001). HBsAg was reported positive in 111/5255 (2.11%) and anti-HCV in 26/2062 (1.26%) tested cases. The age-adjusted prevalence was estimated to be 2.39% for HBV and 1.79% for HCV. Taking into account individuals at high risk for viral hepatitis not included in the survey, the 'true' prevalence was estimated to be 2.58% for HBV and 1.87% for HCV. Anti-HBV and anti-HCV treatment had been taken by 36/111 (32.4%) chronic HBV and 15/26 (57.7%) chronic HCV patients. In conclusion, almost 50% of chronic HBV and 80% of chronic HCV patients in Greece may be unaware of their infection, while only 32% or 58% of diagnosed chronic HBV or HCV patients, respectively, have been ever treated. Therefore, intensive efforts are required to improve the efficacy of screening for HBV and particularly for HCV as well as to reduce the barriers to treatment among diagnosed patients.

Renal transplantation from hepatitis B surface antigen (HBsAg)-positive donors to HBsAg-negative recipients: a case of post-transplant fulminant hepatitis associated with an extensively mutated hepatitis B virus strain and review of the current literature.

PURPOSE: The purpose of this study was to present a fatal case of fulminant hepatitis B (FHB) that developed in a renal transplant recipient, immunized against hepatitis B, 1 year post transplantation. METHODS: Polymerase chain reaction amplification and full genome sequencing were performed to investigate whether specific mutations were associated with hepatitis B virus (HBV) transmission and FHB. RESULTS: Molecular analysis revealed multiple mutations in various open reading frames of HBV, the most important being the G145R escape mutation and a frameshift mutation-insertion (1838insA) within the pre-C/C reading frame. CONCLUSIONS: Our results highlight the possibility of developing FHB, despite previous immunization against HBV or administration of hyperimmune gammaglobulin, because of the selection of escape virus mutants. The current literature and guidelines regarding renal transplantation from hepatitis B surface antigen (HBsAg)-positive to HBsAg-negative patients were also reviewed.

Searching for West Nile virus (WNV) in Greece.

West Nile virus (WNV), a mosquito-borne flavivirus, has increasingly become a concern in both America and Europe due to its complex and unpredictable lifecycle. Transfusion-associated transmission of the WNV has been well documented during the last few years. This study aimed to detect the presence of WNV in: (i) cerebrospinal fluid (CSF) specimens derived from aseptic meningitis cases in Greece and (ii) Greek blood donations. A total of 115 CSF specimens from patients suffering from aseptic meningitis and 9590 blood samples were collected from seven Greek hospitals during the periods June to October 2006 and 2007 and tested for investigational purposes. Both blood and CSF samples were tested for the presence of WNV RNA by using the PROCLEIX WNV assay. None of 115 CSF and 9590 blood donor samples was found positive according to our testing algorithms. Despite the presence of WNV in Balkan countries, WNV has not reached significant levels in Greece.

Determinants of acquisition and carriage of Staphylococcus aureus in infancy.

Nasal carriage of Staphylococcus aureus is a major risk factor for invasive S. aureus disease. The aim of this study was to define factors associated with carriage. We conducted a prospective, longitudinal community-based study of infants and their mothers for a period of 6 months following delivery. The epidemiology of carriage was examined for 100 infant-mother pairs. Infant carriage varied significantly with age, falling from 40 to 50% during the first 8 weeks to 21% by 6 months. Determinants of infant S. aureus carriage included maternal carriage, breastfeeding, and number of siblings. Bacterial typing of S. aureus was performed by pulsed-field gel electrophoresis and multilocus sequence typing. The majority of individuals carried a single strain of S. aureus over time, and the mother was the usual source for colonizing isolates in infants. The effect of other components of the normal nasal flora on the development of S. aureus carriage was examined in 157 consecutive infants. Negative associations (putative bacterial interference) between S. aureus and other species occurred early in infancy but were not sustained. An increasing antistaphylococcal effect observed over time was not attributable to bacterial interference. S. aureus carriage in infants is likely to be determined by a combination of host, environmental, and bacterial factors, but bacterial interference does not appear to be an ultimate determinant of carrier status.

Evidence for lack of cross-genotype protection of CD4+ T cell responses during chronic hepatitis C virus infection.

CD4+ T lymphocyte responses are thought to play a major role in control of the hepatitis C virus (HCV). Few, however, have been mapped down to the level of peptide and HLA restriction. Furthermore, the ability of such T cells to respond to viruses which differ in genotype has not been addressed in detail. In most cases of persistent infection with HCV, CD4 proliferative responses are weak or absent. From a large cohort of persistently infected patients, we identified an individual with unusually robust and persistent responses in the face of chronic infection. We firstly mapped two peptide epitopes to regions of the nonstructural protein NS4 (aa1686-1705 and aa 1746-1765). However, in contrast to the genotype 1a derived antigens used for mapping, the infecting virus was identified as genotype 3a. Strikingly, the patient's CD4 response to these epitopes were specific only for the genotype 1a sequence, and did not recognize genotype 3a synthetic peptides. Serologic assays indicated that prior exposure to HCV of genotype 1 had occurred. This patient therefore maintains strong CD4 proliferative responses which are genotype specific and not cross-reactive. The apparent 'misdirection' of these nonprotective responses has important implications for the role of natural and vaccine induced CD4 responses in the face of variable viruses.

The ica operon and biofilm production in coagulase-negative Staphylococci associated with carriage and disease in a neonatal intensive care unit.

Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in the neonatal intensive care unit (NICU). We evaluated the hypothesis that the ica operon and biofilm production are associated with CoNS disease in this setting. CoNS associated with bacteremia or blood culture contamination and from the skin of infants with CoNS bacteremia or healthy controls were obtained during a prospective case-control study on a busy NICU. A total of 180 strains were identified, of which 122 (68%) were Staphylococcus epidermidis and the remainder were S. capitis (n = 29), S. haemolyticus (n = 11), S. hominis (n = 9), S. warneri (n = 8), and S. auricularis (n = 1). The presence of the genes icaA, icaB, icaC, and icaD was determined by PCR, and biofilm production was examined using qualitative (Congo red agar [CRA]) and quantitative (microtiter plate) techniques. There were no significant differences in the presence of the ica operon or CRA positivity among the four groups of strains. However, quantitative biofilm production was significantly greater in strains isolated from either the blood or the skin of neonates with S. epidermidis bacteremia. We conclude that the quantity of biofilm produced may be associated with the ability to cause CoNS infection. This conclusion suggests that the regulation of biofilm expression may play a central role in the disease process.

Fibronectin-binding protein A of Staphylococcus aureus has multiple, substituting, binding regions that mediate adherence to fibronectin and invasion of endothelial cells.

Invasive Staphylococcus aureus infection frequently involves bacterial seeding from the bloodstream to other body tissues, a process necessarily involving interactions between circulating bacteria and vascular endothelial cells. Staphylococcus aureus fibronectin-binding protein is central to the invasion of endothelium, fibronectin forming a bridge between bacterial fibronectin-binding proteins and host cell receptors. To dissect further the mechanisms of invasion of endothelial cells by S. aureus, a series of truncated FnBPA proteins that lacked one or more of the A, B, C or D regions were expressed on the surface of S. aureus and tested in fibronectin adhesion, endothelial cell adhesion and invasion assays. We found that this protein has multiple, substituting, fibronectin-binding regions, each capable of conferring both adherence to fibronectin and endothelial cells, and endothelial cell invasion. By expressing S. aureus FnBPA on the surface of the non-invasive Gram-positive organism Lactococcus lactis, we have found that no other bacterial factor is required for invasion. Furthermore, we have demonstrated that, as with other cell types, invasion of endothelial cells is mediated by integrin alpha5beta1. These findings may be of relevance to the development of preventive measures against systemic infection, and bacterial spread in the bacteraemic patient.

Ex vivo analysis of phenotype and TCR usage in relation to CD45 isoform expression on cytomegalovirus-specific CD8+ T lymphocytes.

Human cytomegalovirus (CMV) is a ubiquitous pathogen which sets up a lifelong persistent infection and which can lead to significant disease in the immunosuppressed. The immunological mechanisms controlling CMV in the long term are not defined completely, but CD8+ T lymphocytes are thought to play an important role. Antiviral CD8+ T lymphocytes may exist in very large pools in healthy individuals. Although the detailed composition of these pools is not completely understood, there is known to be heterogeneity, in particular of CD45 isoform expression. We have therefore investigated the CD8+ T-lymphocyte response against CMV directly ex vivo using Class I tetramers combined with stains for a range of phenotypic markers followed by four-colour flow cytometric analysis. In particular, we examined expression of these phenotypic markers in relation to the expression of CD45 isoforms. We found that a spectrum of phenotypes exists stably, from CD45R0(high)/RA(low) through CD45RA(high)/R0(low), and that expression of other surface markers such as CD28 and CD62L, and also TCR usage, may vary in parallel with CD45 isoform expression. In some individuals, expansions of antigen-specific CD8+ T lymphocytes bearing specific TCR Vbeta chains were restricted to cells of particular CD45 isoforms. Immunity against CMV comprises a large population of CD8+ T lymphocytes with heterogeneous potential, a spectrum in which CD45 isoform expression may play a central role.

Studies of human antiviral CD8+ lymphocytes using class I peptide tetramers.

Understanding the interactions between a host and a pathogen relies crucially on quantitative measurements of immune responses. Until recently, measurements of the levels of cellular immune responses, i.e. those mediated by CD4+ and CD8+ T lymphocytes have depended largely on culture in vitro and subsequent measurement of specific functions (such as cytolysis). More recently, new technologies based around tetrameric class I peptide complexes (tetramers) have allowed immunologists to measure CD8+ T lymphocyte levels directly ex vivo and independently of function. Since CD8+ lymphocytes play a key role in a number of important human viral infections, these tools have yielded useful insights into the dynamics, phenotype and function of human antiviral lymphocyte populations. In this review we describe some of the basic aspects of the biology of virus-specific CD8+ lymphocytes, and the current methods available to detect them. The use of tetramers has, in just four years, transformed our understanding of the immune responses against HIV, HTLV-1, HBV, HCV, CMV and EBV, and holds promise in a number of areas where quantitative analysis of the antiviral response in terms of both number and function is critical.

Direct ex vivo measurement of CD8(+) T-lymphocyte responses to human parvovirus B19.

Parvovirus B19 is a common human pathogen which can cause severe syndromes, including aplastic anemia and fetal hydrops. The mapping of the first parvovirus B19-derived CD8(+) T-lymphocyte epitope is described. This HLA-B35-restricted peptide derives from the nonstructural (NS1) protein and is strongly immunogenic in B19 virus-seropositive donors.

A multi-centre study of nosocomial methicillin-resistant Staphylococcus aureus in Greece. Greek MRSA Study Group.

All 105 non-replicate consecutive Staphylococcus aureus strains isolated in 1997 from seven Greek hospitals, were found to be susceptible to vancomycin, teicoplanin and chloramphenicol, but only five (8%) were susceptible to all 16 antibiotics tested. Forty-three (41%) isolates were methicillin-resistant, 58% homogeneously (homMRSA) and 42% heterogeneously (hetMRSA). Resistance of homMRSA strains to other antibiotics was generally high (88-100%), although only one strain was resistant to netilmicin. Resistance in hetMRSA (6-39%) or in MSSA (5-11%) was significantly lower. Consequently, the majority (76%) of homMRSA were multi-drug resistant, while the dominant phenotype of hetMRSA and MSSA was resistance to penicillin (50% and 76%, respectively). Comparison of these strains with isolates from 1994 showed higher resistance rates to erythromycin among MSSA, to erythromycin and amikacin among hetMRSA and to rifampicin among homMRSA strains.

Reduced susceptibility to vancomycin of nosocomial isolates of methicillin-resistant Staphylococcus aureus.

The MICs of vancomycin for 56 random nosocomial Staphylococcus aureus isolates homogeneously resistant to methicillin (homMRSA), 16 heterogeneously resistant isolates (hetMRSA) and 25 susceptible isolates (MSSA) were determined by a standard broth microdilution method. Representative isolates were also tested by an agar incorporation method, the Etest and population analysis. Although always in the susceptible range, MICs of vancomycin for homMRSA were significantly higher than those for hetMRSA or MSSA. Moreover, a homMRSA strain belonging to one of the major Greek MRSA clones contained a sub-population of cells that could grow in the presence of vancomycin 8 mg/L at a frequency of 6.7 x 10(-8).

Streptococcus agalactiae: a vaginal pathogen?

The significance of Streptococcus agalactiae as an aetiological agent in vaginitis was evaluated. A total of 6226 samples from women who presented with vaginal symptoms was examined. The presence of >10 leucocytes/high-power field (h.p.f.) was taken to be the criterion of active infection. S. agalactiae was isolated from 10.1% of these samples. The isolation rates of other common pathogens such as Candida spp., Gardnerella vaginalis and Trichomonas spp. were 54.1%, 27.2% and 4.2%, respectively, in the same group of patients. In contrast, the isolation rates of these micro-organisms in the group of patients who had no infection (<10 leucocytes/h.p.f.) were 4.2%, 38.3%, 33% and 0.5%, respectively. In the majority of samples from which S. agalactiae was isolated, it was the sole pathogen isolated (83%) and its presence was associated with an inflammatory response in 80% of patients. Furthermore, the relative risk of vaginal infection with S. agalactiae (2.38) in patients with purulent vaginal discharge was greater than that of Candida spp. infection (1.41) and lower than that of Trichomonas spp. infection (8.32). These data suggest that S. agalactiae in symptomatic women with microscopic evidence of inflammation should be considered a causative agent of vaginitis.