Value of adding an IGRA to the TST to screen for latent tuberculous infection in Greek health care workers.

SETTING: Ioannina University Hospital, Ioannina, Greece. OBJECTIVE: To evaluate the value of adding an interferon-gamma release assay (IGRA) to the tuberculin skin test (TST) for detecting latent tuberculous infection (LTBI) in a Greek university hospital among health care workers (HCWs) predominantly vaccinated with bacille Calmette-Guérin (BCG). DESIGN: Of 788 HCWs enrolled, 68.1% were BCG-vaccinated. A TST ⩾ 10 mm was considered positive and was followed by the QuantiFERON-TB(®) Gold In-Tube assay (QFT-GIT) in a two-step strategy. RESULTS: Of the enrolled HCWs, 36.4% were TST-positive, of whom only 14.4% were IGRA-positive. Agreement between the tests was poor (κ = 0.019; 95%CI -0.014-0.05, P = 0.355). Both TST and IGRA positivity increased with TST diameter, from 5.7% in TST 10-14 mm to 48.8% in TST ⩾20 mm. TST-positive, IGRA-negative results were most likely in younger, recently BCG-vaccinated HCWs (84.6% in those aged 20-29 years) and less likely in older HCWs (45% in those aged 50-59 years). The two-step strategy would have been more cost saving compared to the TST-only approach if adherence to LTBI treatment in our cohort had been ⩾24%. CONCLUSIONS: Poor overall agreement between TST and QFT-GIT was found. Use of IGRA as a second step in TST-positive cases offers an appropriate tool for LTBI detection among BCG-vaccinated HCWs in low-TB-incidence settings.

A 12-year survey of methicillin-resistant Staphylococcus aureus infections in Greece: ST80-IV epidemic?

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) infections. Severe MRSA infections have been associated with the virulence factor Panton-Valentine leukocidin (PVL). The aim of this study was to investigate susceptibility patterns, the presence of toxin genes, including that encoding PVL, and clonality among MRSA isolates collected from patients in Greece over a 12-year period. MRSA isolates were collected from January 2001 to December 2012 from six different hospitals. Antibiotic susceptibility was determined with the disk diffusion method and the Etest. The presence of the toxic shock syndrome toxin-1 gene (tst), the enterotoxin gene cluster (egc) and the PVL gene was tested with PCR. The genotypic characteristics of the strains were analysed by SCCmec and agr typing, and clonality was determined with pulsed-field gel electrophoresis and multilocus sequence typing. An increasing rate of MRSA among S. aureus infections was detected up to 2008. The majority of PVL-positive MRSA isolates belonged to a single clone, sequence type (ST)80-IV, which was disseminated both in the community and in hospitals, especially during the warmest months of the year. Carriage of tst was associated with ST30-IV, whereas egc was distributed in different clones. CA-MRSA isolates were recovered mainly from skin and soft tissue infections, whereas HA-MRSA isolates were associated with surgical and wound infections. During the period 2001-2012, ST80-IV predominated in the community and infiltrated the hospital settings in Greece, successfully replacing other PVL-positive clones. The predominance of ST239-III in HA-MRSA infections was constant, whereas new clones have also emerged. Polyclonality was statistically significantly higher among CA-MRSA isolates and isolates from adult patients.

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.

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.

Serologic prevalence of coxsackievirus group B in Greece.

Coxsackieviruses are human enteroviruses, which have been associated with myocarditis/pericarditis and sudden death. In one investigation (Spanakis N, Manolis EN, Tsakris A, Tsiodras S, Panagiotopoulos T, Saroglou G, and Legakis NJ: J Clin Pathol 2005;58:357-360), a cluster of cases of fatal myocarditis in Greece was linked to coxsackievirus B3. The information from this investigation prompted us to study serologically the prevalence of coxsackieviruses B throughout Greece. Sera were obtained from 506 healthy blood donors from various transfusion centers, covering the entire country. All sera were tested for the presence of IgG and IgM antibodies, using ELISAs with various antigenic specificities: (1) heat-denatured coxsackievirus type B1 and B5 virions, (2) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B3, and (3) a synthetic peptide from the N terminus of the VP1 protein of coxsackievirus B4. Sera positive for IgG antibodies against coxsackieviruses B1/B5, B3, and B4 were detected in 6.7 to 21.6% of the individuals tested in the various regions of Greece. Statistical analysis revealed that the highest prevalence of IgG antibodies against coxsackieviruses B1/B5 was found in blood donors from Crete (p = 0.025), whereas the highest prevalence against coxsackievirus B4 was detected in blood donors from Athens (p = 0.01). IgM antibodies against coxsackievirus B were detected at low percentage, less than 5%, with no significant viral preference for particular geographic regions. The preference of anti-coxsackievirus IgG antibodies for particular geographic regions could be potentially related to the previously reported clustering of cases of insulin-dependent diabetes mellitus and myocarditis in Athens and Crete, respectively.

Use of mutational pattern in 5'-NCR and VP1 regions of polioviruses for molecular diagnosis.

Polioviruses are members of the enterovirus genus, belonging to the Picornaviridae family. They are the causative agents of poliomyelitis, a paralytic and sometimes fatal disease in humans. The number of poliomyelitis cases caused by wild poliovirus infections has been dramatically reduced by the extensive use of two available vaccines: the inactivated poliovirus vaccine (IPV) and the oral poliovirus vaccine (OPV). Despite the importance of OPV in the reduction of poliomyelitis cases, one of the disadvantages associated with this vaccine is the rare occurrence of vaccine-associated paralytic poliomyelitis (VAPP) in vaccinees or their healthy contacts through the accumulation of mutations and/or recombination in Sabin strains genome. Thirteen clinical isolates originating from healthy vaccinees and VAPP cases were investigated in order to identify genomic modifications in 5' non-coding region (5'-NCR) and VP1 genomic regions. The analysis of samples was conducted by RT-PCR, RFLP, sequencing and bioinformatics analysis. All clinical isolates were characterized as OPV-like viruses. Our results showed that analysis of 5'-NCR and VP1 regions of Poliovirus Sabin strains is important in order to identify mutations that increase the neurovirulence conducting to the eventuality of emergence of VAPP cases.

Conventional and molecular investigation of meningococcal isolates in relation to two outbreaks in the area of Athens, Greece.

Two local outbreaks caused by serogroup B Neisseria meningitidis occurred in the Athens area of Greece during 2003. In total, 30 N. meningitidis isolates from patients and carriers, as well as sporadic cases, were investigated by conventional techniques (serogroup, serotype and serosubtype), multilocus sequence typing (MLST), analysis of variable number tandem repeats (VNTR) and random amplified polymorphic DNA (RAPD) analysis. Compared with the two other molecular techniques, VNTR analysis was a simple, reliable and highly discriminatory method for fine typing of meningococcal isolates, showing a good correlation with the epidemiological data for the two outbreaks analysed.

The effect of short-time microwave exposures on Escherichia coli O157:H7 inoculated onto chicken meat portions and whole chickens.

Small portions of fresh chicken breasts weighting 20 g each and fresh whole chickens, weighting on average 1310 g each, were inoculated with Escherichia coli O157:H7 (10(5)-10(6) cfu/g) and cooked, using two different domestic microwave ovens at full power. The chicken portions were heated for 5, 10, 15, 20, 25, 30, and 35 s and the whole chickens for 22 min. Following exposures, viable counts and temperature measurements were performed. Although the chicken breast portions looked well-cooked after 30 s of MW heating at a mean end-point surface temperature of 69.8 degrees C, a mean concentration of 83 cfu/g E. coli O157:H7 cells was recovered. Elimination of E. coli O157:H7 cells occurred only after 35 s of MW exposure at 73.7 degrees C. When whole chickens were thoroughly cooked by MW heating, the final subsurface temperatures, measured in the thighs and wings, ranged from 60.2 degrees C to 92 degrees C and viable cells of E. coli O157:H7 were recovered from all samples of whole chicken. The results indicate that short time exposures of chicken portions to microwave heating do not eliminate E. coli O157:H7.

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.

Membrane adsorption with direct cell culture combined with reverse transcription-PCR as a fast method for identifying enteroviruses from sewage.

We present a new approach for the detection and identification of enteroviruses concentrated and isolated from sewage. Samples were collected from two study sites located at Nicosia and Limassol sewage treatment plants in Cyprus. Viruses were adsorbed to cellulose nitrate membrane filters, cultured directly from the membrane filters by using the VIRADEN method, and identified by reverse transcription-PCR, followed by 5' untranslated region (5'-UTR) restriction fragment length polymorphism (RFLP) analysis and partial sequencing of the VP1 protein coding region. Initial subgrouping based on the HpaII restriction profile showed that all of the isolates except one belonged to the same genetic subcluster. Partial VP1 sequencing revealed that most isolates belonged to serotypes coxsackie B4 (42.5%) and coxsackie Alpha9 (30%), whereas coxsackie B2 (17.5%) and coxsackie B1 (3%) isolates were less frequently observed. One poliovirus type 2 isolate (2.5%) of vaccine origin was also found. The HpaII digests predicted the genetic subcluster for all isolates. They also accurately differentiated the isolates as nonpolio or polio isolates. This approach seems to be very promising for environmental surveillance of enterovirus circulation and epidemiology, with all of the significant effects that this entails for public health. Partial VP1 sequencing is efficient for molecular serotyping of enteroviruses, while 5'-UTR RFLP analysis with HpaII can also be considered an asset for the initial subclassification of enterovirus isolates.

Nosocomial lower respiratory tract infections: prevalence and risk factors in 14 Greek hospitals.

Nosocomial lower respiratory tract infections (NLRTIs) are associated with significant morbidity and mortality. The aim of this study was to investigate the epidemiological features of NLRTIs in Greece, where knowledge about these infections is limited. Two point-prevalence studies of hospital-acquired infections were carried out in 14 Greek hospitals located throughout the country, one in 1999 and one in 2000. NLRTIs were diagnosed in accordance with the Centers for Disease Control and Prevention (CDC) definitions. Among the 7,120 hospitalized patients registered during the two studies, 610 (8.6%) cases of hospital-acquired infections were identified, of which 200 (32.8%) were NLRTIs. Sixty-nine (34.5%) patients had pneumonia, and the remaining 131 (65.5%) patients had bronchitis. The greatest prevalence of NLRTI was found in the adult ICUs (30.4%). Male gender, age >65 years, mechanical ventilation, tracheostomy, an intravenous central line, and an indwelling urethral catheter were the main risk factors. There was no significant difference in the incidence of NLRTI among hospital-acquired infections between the 1999 study and the 2000 study. The causative microorganism was identified in 78 of 200 (39%) cases, and 103 strains were isolated. The majority of strains (67%) were gram-negative bacteria. The most frequently isolated microorganisms were Pseudomonas aeruginosa (22.3%), Acinetobacter spp. (19.4%), Klebsiella pneumoniae (12.6%), and Staphylococcus aureus (10.7%). There was no difference between the two prevalence studies in the frequency of isolation of the microorganisms. NLRTI was the leading cause of morbidity and mortality among hospitalized patients with hospital-acquired infections in Greek hospitals. Gram-negative microorganisms were the most frequently isolated pathogens.

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.

Isolation of a rare Escherichia coli O157:H7 strain from farm animals in Greece.

A strain of Escherichia coli O157:H7 was isolated from goat faeces during a surveillance study on the prevalence of this serotype of E. coli in farm animals in Greece. Three hundred and fifty one faecal samples were collected from goat, sheep and cattle breeding farms in the area of Epirus, Northwestern Greece. The E. coli O157:H7 isolate was nonsorbitol-fermenter, produced only VT2 and showed a beta-glucuronidase positive activity, a rather unusual biochemical feature for the E. coli O157:H7 serotype. No other strain of E. coli O157:H7 was isolated from the faecal samples of the rest farm animals examined, thus the overall prevalence of animal carriage was found to be 0.2%. The findings also indicate that goats can be a reservoir of E. coli O157:H7 and goat milk, dairy products and meat may serve as a vehicle for the pathogen transmission to humans.

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.

Isolation of Escherichia coli O157:H7 from foods in Greece.

The presence of Escherichia coli O157:H7 in various foods of animal origin was surveyed in northwestern Greece. Six hundred samples of unpasteurized cows', ewes' and goats' milk, raw minced meat, uncooked frozen beef hamburgers, sandwiches (containing ham or turkey, mixed vegetable salad with mayonnaise and lettuce), fresh traditional Greek pork sausages and swine intestines appropriate for traditional Greek kokoretsi were assayed for E. coli serogroup O157:H7 using the standard cultural method and the immunomagnetic separation technique. The pathogen was detected in 1 out of 100 (1.0%) samples of ewes' milk, 1 out of 75 (1.3%) fresh sausages and 1 out of 50 (2.0%) swine intestines prepared for kokoretsi. The isolated strains were nonsorbitol fermenters, MUG-negative, O157 agglutinating, verotoxin-producing and carried both VT1 and VT2 genes. The three isolated strains were tested for antibiotic resistance and were found to be susceptible to eight antimicrobial agents (ampicillin, chloramphenicol, kanamycin, nalidixic acid, norfloxacin, streptomycin, sulfamethoxazole-trimethoprim and tetracycline).

Prevalence study of hospital-acquired infections in 14 Greek hospitals: planning from the local to the national surveillance level.

A prevalence study of hospital-acquired infections (HAI) was carried out in 14 of 112 Greek hospitals (15.7%), scattered throughout Greece. Five of seven Greek university hospitals and nine regional hospitals participated in the one-day study, and 3925 hospitalized patients (10.5% of the total hospital beds in Greece) were recorded. The aim of this project was to organize a surveillance of HAI with the participation of the greatest possible number of Greek hospitals, transferring the experience from the local Cretan infection control network in an effort to create a nationwide network. Special attention was paid to recruit all Greek university hospitals in our attempt to expand the study base. Co-ordination of the participating centres, education of the infection control teams on surveillance methods, preparation of agreed definitions, and elaboration of the protocol for the collection of the data were the major objectives of this study. The difficulties, however, were limited resources and the lack of skilled personnel. The overall prevalence of HAI was found to be 9.3%. The most common HAI recorded involved lower respiratory tract infections (30.3%), followed by urinary tract infections (22.7%), bloodstream infections (15.8%), and surgical site infections (14.8%). The greatest prevalence rate was found in the adult ICU (48.4%), followed by the neonatal ICU (30.3%). The duration of hospitalization, the number of operations, the total number of used devices and invasive procedures were significantly correlated with HAI. Positive cultures were found in 51.5% of the cases. The most frequently isolated micro-organisms were: Pseudomonas aeruginosa (16.6%), Escherichia coli (10.8%), Klebsiella pneumoniae (10.3%), Staphylococcus epidermidis (8.1%) and Staphylococcus aureus (7.6%). The administration of antibiotics was also recorded. The prevalence of antibiotic use was 51.4%.