Constantina Gartzonika

An eternal microbe: Brucella DNA load persists for years after clinical cure.

BACKGROUND Despite the continuing high incidence of brucellosis, vague aspects of pathophysiology, diagnosis, and treatment continue to exist, particularly with regard to the ability of Brucella species to survive inside the host. METHODS A quantitative real-time polymerase chain reaction assay was used for monitoring bacterial DNA load in brucellosis-affected patients throughout different disease stages. Three or more specimens per patient were obtained (1 at diagnosis, 1 at the end of treatment, and at least 1 during the follow-up period) from 39 patients with acute brucellosis. RESULTS The majority of patients (87% at the end of treatment, 77% at 6 months after treatment completion, and 70% at >2 years after treatment) exhibited persistent detectable microbiological load despite being asymptomatic. The 3 patients who experienced relapse did not exhibit any statistically significant difference in their bacterial load at any stage of disease or during follow-up. CONCLUSION Brucella melitensis DNA persists despite appropriate treatment and apparent recovery. This finding offers a new insight into the pathophysiology of the disease: B. melitensis is a noneradicable, persisting pathogen.

The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece

OBJECTIVES Despite the fact that the NDM-1 carbapenemase has successfully disseminated worldwide, outbreaks remain uncommon in the European region. We describe the characteristics of the first outbreaks caused by NDM-1-producing Klebsiella pneumoniae clonal isolates in Greece. METHODS Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant Enterobacteriaceae isolates, which gave a positive modified Hodge test and were phenotypically suspected of metallo-β-lactamase production, were recovered from patients hospitalized at Ioannina University Hospital. Resistance genes were identified by PCR and sequencing. Plasmid profiling, conjugation experiments, enterobacterial repetitive intergenic consensus PCR, PFGE and multilocus sequence typing (MLST) were performed. Patient records were retrieved to access patterns of acquisition. RESULTS Molecular testing verified the presence in 78 K. pneumoniae isolates, collected from 71 patients, of the blaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was located on a narrow host range IncFII-type plasmid, of ∼95 kb, flanked upstream by a non-truncated ISAba125 element and downstream by the bleMBL gene. Genotyping clustered all K. pneumoniae isolates into a single clonal type with one subtype and MLST assigned them to sequence type 11. Two outbreaks were noted, the first between November and December 2011 involving four patients and the second initiated in May 2012 and ongoing, involving the remaining patients. All but two cases were characterized as hospital acquired. No links to immigration or travel history to endemic areas were established. CONCLUSIONS This survey highlights the successful undetected dissemination of yet another carbapenemase in Greece and strengthens the hypothesis of a latent NDM-1 cluster in the Balkan region.

Effects of rosuvastatin with or without ezetimibe on clinical outcomes in patients undergoing elective vascular surgery: results of a pilot study.

Objective: Cardiovascular complications represent a major cause of morbidity and mortality in patients undergoing vascular surgery. This was a prospective randomized, open-label study to investigate the effect of lipid-lowering treatment by statin monotherapy or intensified by combining statin with ezetimibe on a 12-month prognosis after vascular surgery. Methods: Patients were randomly assigned to receive rosuvastatin (RSV) 10 mg/d or rosuvastatin 10 mg/d plus ezetemibe (RSV/EZT) 10 mg/d, starting prior to scheduled surgical procedure. The primary end point was the first major cardiovascular event, including death from cardiac causes, nonfatal myocardial infarction, ischemic stroke, and unstable angina. Results: A total of 136 patients assigned to RSV and 126 to RSV/EZT completed the study protocol. As many as 6.6% of patients in the RSV group experience a major cardiovascular event within 30 days after surgery versus 5.6% in the RSV/EZT group (P = .72). From month 1 to 12 of the follow-up period, primary end point was observed (9 taking RSV vs 2 in the RSV/EZT group [P = .04]). Intensified lipid-lowering therapy with RSV/EZT was associated with a greater decrease in low-density lipoprotein cholesterol levels compared with RSV (75.87 ± 31.64 vs 87.19 ± 31.7, P = .004), while no differential effect on triglyceride, high-density lipoprotein cholesterol or high-sensitivity C-reactive protein levels was noted between groups. Conclusion: Our findings indicate that statin therapy intensified by ezetimibe may reduce the incidence of cardiovascular events within the first 12 months after vascular surgery. Nonetheless, whether the use of ezetimibe as an add-on therapy to reduce cardiovascular risk in these patients needs to be tested in larger future studies.

Usefulness of Herpes Consensus PCR methodology to routine diagnostic testing for herpesviruses infections in clinical specimens.

The purposes of the study were to assess the usefulness of simultaneously amplifying herpes simplex virus 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus and human herpesvirus 6 DNA in various clinical specimens and to analyze clinical events in patients presenting positive results. A total of 763 clinical samples obtained from 758 patients, including 115 cerebrospinal fluids, 102 aqueous fluids, 445 swabs from genital (152), oro-facial (138) and other (155) skin lesions, 96 eye swabs and 5 bronchoalveolar lavages, were tested by using the Consensus polymerase chain reaction methodology. The clinical files of the patients were consulted retrospectively. 171 of the 758 patients (22.5%) were positive for at least one of the six target viruses: herpes simplex virus 1 (n = 95), varicella-zoster virus (n = 40), herpes simplex virus 2 (n = 21), herpes simplex virus 1 plus herpes simplex virus 2 (n = 8), cytomegalovirus (n = 4), Epstein-Barr virus (n = 1), human herpesvirus 6 (n = 1), and herpes simplex virus 1 plus human herpesvirus 6 (n = 1). The Consensus methodology enabled the rapid and accurate detection of herpesviruses in various clinical specimens and provided a reliable tool in the diagnosis of herpetic infections.

Postoperative levels of cardiac troponin versus CK-MB and high-sensitivity C-reactive protein for the prediction of 1-year cardiovascular outcome in patients undergoing vascular surgery

ObjectiveThis study evaluated comparatively the predictive value of postoperative cardiac troponin I (cTnI), creatinine kinase (CK)-MB, and high-sensitivity C-reactive protein (hs-CRP) in 1-year cardiovascular mortality and morbidity in patients undergoing elective vascular surgery. MethodsA total of 295 consecutive patients undergoing elective noncardiac vascular surgery were prospectively followed-up over a period of 12 months. The levels of cTnI, CK-MB, and hs-CRP were measured preoperatively and 24 h after operation. The primary endpoint was the composite of cardiovascular death, nonfatal acute myocardial infarction, ischemic stroke, and unstable angina. ResultsThe primary endpoints occurred in 11 patients (3.8%). Receiver operating characteristic curve analysis showed that postoperative cTnI was a strong predictor of a cardiovascular event during 1-year follow-up (area under the curve, 0.852; P<0.001). Areas under the curve for hs-CRP and for CK-MB were 0.734 (P=0.008) and 0.494 (P=0.947). A threshold cTnI value of 0.4 ng/ml was highly associated with the occurrence of a cardiovascular event, with a sensitivity of 80% and specificity of 81%. Furthermore, cTnI levels provided a significantly better prediction than CK-MB levels (P=0.009) and tended to be superior to hs-CRP (P=0.2). ConclusionPostoperative cTnI levels seem to be superior to CK-MB and hs-CRP levels for the prediction of 1-year cardiovascular mortality and morbidity in patients undergoing elective vascular surgery.

Molecular characterization of a new intergenotype Norovirus GII recombinant.

Human noroviruses (NoVs) of the Caliciviridae family are a major cause of epidemic gastroenteritis. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In the present study, we report the development of a broadly reactive RT-PCR assay, which allowed the characterization of strain A6 at molecular level, established its genetic relationship at the sub-genogroup level and classified A6 strain at the sub-genotype level. The detection was carried out initially by enzyme-linked immunosorbent assay (ELISA) and the subsequent detection and molecular characterization of NoV strain was achieved by reverse transcription-PCR and sequencing. Based on the sequence analysis, A6 strain was revealed to belong to the GII genogroup of NoVs. Partial ORF1 gene sequencing analysis and complete ORF2 gene sequencing revealed that ORF1 and ORF2 belonged to two distinct genotypes GII/9 and GII/6, respectively, making obvious that A6 strain is a rare intergenotypic recombinant within the genogroup GII between GII.9 and GII.6 genotypes. A6 strain represents the first human NoV from Greece, whose genome has been partially (ORF1&ORF3) and completed (ORF2) sequenced. To our knowledge the recombination event GII.9/GII.6 in RdRp and capsid gene, respectively, that was revealed in the present study is reported for the first time.

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006.

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C–3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002–2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.

Characterization of novel intergenogroup and intergenotype recombinant noroviruses from central Greece.

Noroviruses (NoVs) are a major causative agent of acute gastroenteritis in humans. They are members of the Caliciviridae family and based on the genetic analysis of the RdRp and capsid regions, human NoVs are divided into three genogroups (Gs), GI, GII, and GIV. The three genogroups further segregate into distinct lineages called genotypes. The NoV genus is genetically diverse and recombination of viral RNA is known to depend upon various immunological and intracellular constraints that may allow the emergence of viable recombinants. In this study, three Noroviral strains detected in clinical samples revealed two hitherto unobserved recombination events between GII.9/GII.4 and GII.9/GI.7 genogroups. To our knowledge, these intergenotype and intergenogroup recombination events of GII.9/GII.4 and GII.9/GI.7, in ORF1 and ORF2 genes respectively are reported for the first time and highlight the ongoing evolution of noroviruses.

Molecular epidemiology and evolutionary dynamics of Echovirus 3 serotype

Echovirus 3 (E3) serotype has been related with several neurologic diseases, although it constitutes one of the rarely isolated serotypes, with no report of epidemics in Europe. The aim of the present study was to provide insights into the molecular epidemiology and evolution of this enterovirus serotype, while an E3 strain was isolated from sewage in Greece, four years after the initial isolation of the only reported E3 strain in the same geographical region. Phylogenetic analysis of the complete VP1 genomic region of that E3 strain and of those available in GenBank suggested three main genogroups that were further subdivided into seven subgenogroups. Further evolutionary analysis suggested that VP1 genomic region of E3 was dominated by purifying selection, as the vast majority of genetic diversity presumably occurred through synonymous nucleotide substitutions and the substitution rate for complete and partial VP1 sequences was calculated to be 8.13×10(-3) and 7.72×10(-3) substitutions/site/year respectively. The partial VP1 sequence analysis revealed the composite epidemiology of this serotype, as the strains of the three genogroups presented different epidemiological characteristics.

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.