Detection of Chlamydia trachomatis inside spermatozoa using flow cytometry: Effects of antibiotic treatment (before and after) on sperm count parameters.

There is increasing evidence that Chlamydia trachomatis (CT) infection can directly affect male fertility. However, only few have investigated the effects of CT on semen parameters, and mostly with inconclusive results. The main aims of this study were to identify CT inside spermatozoa, and the possible pre and post antibiotic treatment effects on the overall semen parameters. We developed a flow cytometric method for the detection of CT inside spermatozoa (SPI™). Briefly, sperm cells were fixed, membrane permeabilized and DNA was loosened using DNAse. Sperm cells were incubated with a primary monoclonal antibody against CT and with a secondary fluorescent antibody (vs primary), and analysed using a flow cytometer. Of 2415 infertile individuals, 48.61% were found positive for CT. 170 CT+ samples were included in the CT antibiotic treatment study. 78.82% (134/170) of the CT+ showed a significant reduction in the percentage of the iCT infected spermatozoa after the antibiotic treatment; 59.70% (80/134) decreased to non-detectable levels. Spermcount data were also recorded. Spermatozoa morphology (normal and teratozoospermia index, TZI) and motility (fast progressive and non-progressive spermatozoa) were statistically significant altered in CT+ pre-treatment vs control group. CT antibiotic treatment showed statistically significant effects on normal spermatozoa morphology, mid-piece and tail defects, and TZI. The study demonstrated that semen flow cytometric analysis of semen could be a valuable tool for faster and accurate identification of individuals with asymptomatic CT infection. It also identified a positive effect of antibiotic therapy on semen parameters, that could help males with infertility.

Differential Expression of microRNAs in Acute and Chronic Heart Failure.

BACKGROUND: MicroRNAs modify protein expression at the post-transcriptional level, and their circulating levels may help identify the underlying molecular pathways. OBJECTIVE: The purpose of this study was to assess the differential expression of microRNAs related to myocardial cell energy substrate, autophagy, and ischaemia in chronic and acute heart failure (HF). METHODS: In this case-control study, we studied 19 patients with acute HF (AHF) and 19 patients with chronic HF (CHF). Basic demographic and clinical characteristics were collected from the patients upon arrival, at 48 hours, and at 120 hours. Blood samples for microRNAs measurements (miR-22, -92a, and -499), B-type natriuretic peptide (BNP), C reactive protein, and high sensitivity cardiac troponin I, were collected at all study points. In this study, we included subjects with a left ventricular ejection fraction of <40%. RESULTS: At baseline, circulating miR-22 levels were 1.9-fold higher (p<0.001), miR-92a levels were 1.25-fold higher (p=0.003), and miR-499 were 5-times lower (p<0.001) in AHF compared to CHF. Interestingly, circulating miR-499 was found to be associated with BNP levels (r=0.47, p=0.01). At follow-up, there was a stepwise increase in the levels of all three examined microRNAs (miR-22, p=0.001, miR-92a, p=0.001, and miR-499, p<0.001) for AHF but not for CHF subjects. CONCLUSION: MicroRNAs -22, -92a, and -499 are differentially expressed in chronic and acute HF subjects. MicroRNA signatures are also differentially expressed up to the discharge of the patients. These findings may have important implications for diagnosis, progression, and treatment of patients with chronic and acute heart failure.

Biomarkers of Atrial Fibrillation in Hypertension.

BACKGROUND: Atrial fibrillation (AF) is the most frequently encountered cardiac arrhythmia globally and substantially increases the risk for thromboembolic disease. Albeit, 20% of all cases of AF remain undiagnosed. On the other hand, hypertension amplifies the risk for both AF occurrences through hemodynamic and non-hemodynamic mechanisms and cerebrovascular ischemia. Under this prism, prompt diagnosis of undetected AF in hypertensive patients is of pivotal importance. METHOD: We conducted a review of the literature for studies with biomarkers that could be used in AF diagnosis as well as in predicting the transition of paroxysmal AF to sustained AF, especially in hypertensive patients. RESULTS: Potential biomarkers for AF can be broadly categorized into electrophysiological, morphological and molecular markers that reflect the underlying mechanisms of adverse atrial remodeling. We focused on P-wave duration and dispersion as electrophysiological markers, and left atrial (LA) and LA appendage size, atrial fibrosis, left ventricular hypertrophy and aortic stiffness as structural biomarkers, respectively. The heterogeneous group of molecular biomarkers of AF encompasses products of the neurohormonal cascade, including NT-pro BNP, BNP, MR-pro ANP, polymorphisms of the ACE and convertases such as corin and furin. In addition, soluble biomarkers of inflammation (i.e. CRP, IL-6) and fibrosis (i.e. TGF-1 and matrix metalloproteinases) were assessed for predicting AF. CONCLUSION: The reviewed individual biomarkers might be a valuable addition to current diagnostic tools but the ideal candidate is expected to combine multiple indices of atrial remodeling in order to effectively detect both AF and adverse characteristics of high risk patients with hypertension.

Biomarkers in Atrial Fibrillation and Heart Failure.

Atrial fibrillation (AF) is the most common sustained arrhythmia encountered in clinical practice and an important contributor to cardiovascular morbidity and mortality. Although the exact mechanisms behind AF are not completely elucidated, the underlying pathophysiological changes have been well described. Predisposal factors for AF include the older age, the increased left atrial size, the decreased left atrial function, the presence of heart failure and left ventricular systolic dysfunction and the presence of coronary heart disease or pulmonary or mitral valve disease. In addition to these factors, emerging evidence demonstrate that myocardial strain, fibrosis and inflammation, are associated with AF as well as the pathogenesis of the arrhythmia. The natruretic peptide system including Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP) is indicative of the level of myocardial strain which may predispose to AF. As a result, the aforementioned peptides are increased in AF patients. The levels of myocardial fibrosis biomarkers, such as ST2 and Galectin-3, are elevated suggesting atrial structural abnormalities, while the increased levels of CRP and Interleukin-6 supplement the inflammatory profile of AF patients. Emerging data for the aforementioned biomarkers are discussed in the present review.

Redox State in Atrial Fibrillation Pathogenesis and Relevant Therapeutic Approaches.

BACKGROUND: Myocardial redox state is a critical determinant of atrial biology, regulating cardiomyocyte apoptosis, ion channel function, and cardiac hypertrophy/fibrosis and function. Nevertheless, it remains unclear whether the targeting of atrial redox state is a rational therapeutic strategy for atrial fibrillation prevention. OBJECTIVE: To review the role of atrial redox state and anti-oxidant therapies in atrial fibrillation. METHOD: Published literature in Medline was searched for experimental and clinical evidence linking myocardial redox state with atrial fibrillation pathogenesis as well as studies looking into the role of redoxtargeting therapies in the prevention of atrial fibrillation. RESULTS: Data from animal models have shown that altered myocardial nitroso-redox balance and NADPH oxidases activity are causally involved in the pathogenesis of atrial fibrillation. Similarly experimental animal data supports that increased reactive oxygen / nitrogen species formation in the atrial tissue is associated with altered electrophysiological properties of atrial myocytes and electrical remodeling, favoring atrial fibrillation development. In humans, randomized clinical studies using redox-related therapeutic approaches (e.g. statins or antioxidant agents) have not documented any benefits in the prevention of atrial fibrillation development (mainly post-operative atrial fibrillation risk). CONCLUSION: Despite strong experimental and translational data supporting the role of atrial redox state in atrial fibrillation pathogenesis, such mechanistic evidence has not been translated to clinical benefits in atrial fibrillation risk in randomized clinical studies using redox-related therapies.

Staphylococcus aureus osteoarticular infections in children: an 8-year review of molecular microbiology, antibiotic resistance and clinical characteristics.

PURPOSE: To investigate the clinical, phenotypic and genotypic characteristics of Staphylococcus aureus strains causing osteoarticular infections in a large paediatric series. METHODOLOGY: Medical records of children who were hospitalized with the diagnosis of community-associated S. aureus (CA-SA) osteomyelitis and/or septic arthritis in the two major tertiary paediatric hospitals of Athens during an 8-year period (2007-2015) were reviewed, and S. aureus isolates were analysed regarding antimicrobial resistance, detection of pathogenicity genes and genotyping using SCCmec, agr typing, PFGE and MLST. RESULTS: During the study period, 123 children with CA-SA osteoarticular infections were identified, and methicillin-resistant S. aureus (MRSA) accounted for 44 of these (35.8 %). Children with MRSA infection had a significantly higher admission rate to the ICU (5.7  vs 0 %, P=0.04) and longer duration of hospitalization (21.6 vs 16.7 days, P=0.04). Sixty-eight isolates [42 (methicillin-sensitive S. aureus) MSSA and 26 MRSA] were available for molecular analysis. All MRSA strains were mecA-positive and most carried the SCCmec IV cassette (23/26, 88 %) and belonged to the PFGE type C (24/26, 92.3 %), agr type 3 (24/26, 92.3 %) and the MLST ST80 clone (24/26, 92.3 %). In contrast, MSSA strains showed polyclonality by PFGE and agr typing. Regarding pathogenicity genes, MRSA vs MSSA isolates showed higher detection rates of PVL (96.2 vs 4.8 %, P<0.0001) and fib (80.8 vs 50 %, P=0.02). CONCLUSIONS: In our study a considerable number of S. aureus osteoarticular infections were due to CA-MRSA isolates, most of which belonged to the ST80 clone and had a higher incidence of specific virulence factors, entailing higher ICU admission rates and a longer duration of hospitalization.

Attachment orientations of Greek cancer patients in palliative care. A validation study of the Experiences in Close Relationships scale (ECR-M16).

PURPOSE: Health-care professionals may serve as attachment figures, nevertheless little research has been made in the palliative context. The psychometric properties of the brief ECR-M16 in Greek cancer patients were explored. METHODS: The ECR-M16 was translated into Greek (G-ECR-M16), and was administered to 100 patients before starting palliative care and 7 days later to test its stability. Patients (N=35) also completed the EORTC QLQ-C30 scales. RESULTS: Cronbach's alphas for the discomfort for closeness, anxiety and avoidance scales were 0.871, 0.762, and 0.761, respectively. Test-retest reliability was very satisfactory (p< 0.0005). Factor analysis yielded three factors (58.75% of the variance). Known-groups validity showed that discomfort with closeness had a statistically significant correlation with advanced disease stage (p=0.022). CONCLUSIONS: The G-ECR-M16 is a valid research tool for the attachment patterns' impact in Greek cancer patients.

Medical and Social Aspects of Syphilis in the Balkans from the mid-19th Century to the Interwar.

The current study presents some aspects of syphilis in the Balkan Peninsula from the 19th century until the Interwar. Ever since the birth of modern Balkan States (Greece, Bulgaria, Turkey and Serbia), urbanization, poverty and the frequent wars have been considered the major factors conducive to the spread of syphilis. The measures against sex work and sexually transmitted diseases (STDs) were taken in two aspects, one medical and the other legislative. In this period, numerous hospitals for venereal diseases were established in the Balkan countries. In line with the international diagnostic approach and therapeutic standards, laboratory examinations in these Balkan hospitals included spirochete examination, Wassermann reaction, precipitation reaction and cerebrospinal fluid examination. Despite the strict legislation and the adoption of relevant laws against illegal sex work, public health services were unable to curb the spread of syphilis. Medical and social factors such as poverty, citizen's ignorance of STDs, misguided medical perceptions, lack of sanitary control of prostitution and epidemiological studies, are highlighted in this study. These factors were the major causes that helped syphilis spread in the Balkan countries during the 19th and early 20th century. The value of these aspects as a historic paradigm is diachronic. Failure to comply with the laws and the dysfunction of public services during periods of war or socioeconomic crises are both factors facilitating the spread of STDs.

Current perspectives on tigecycline resistance in Enterobacteriaceae: susceptibility testing issues and mechanisms of resistance.

During the past decades, rates of multidrug-resistant (MDR) and carbapenem-resistant (CR) Enterobacteriaceae clinical isolates, mainly Klebsiella spp., Escherichia coli, Enterobacter spp., Proteus spp. and Serratia marcescens, have increased, considerably restricting effective antimicrobial treatments. Tigecycline, the first member of the glycylcyclines, has been approved by the US Food and Drug Administration (FDA) for the treatment of complicated skin and soft-tissue, complicated intra-abdominal and community-acquired bacterial respiratory infections and is increasingly administered against MDR Enterobacteriaceae. Although resistance has gradually appeared, tigecycline still remains relatively active among Enterobacteriaceae, with resistance rates largely <10% in most wide-scale surveillance studies. Tigecycline resistance has been reported in some studies to be elevated among extended-spectrum ß-lactamase (ESBL)-producing, MDR, extensively drug-resistant and CR isolates. Broth microdilution (BMD) is the reference method for tigecycline susceptibility testing, but disagreements have been reported between the methods applied for routine tigecycline susceptibility testing. Therefore, confirmation of daily tigecycline susceptibility testing with BMD appears important in order to avoid misclassification of isolates. Various mechanisms have been reported to confer tigecycline resistance, with RND-type transporters, mainly the AcrAB efflux pump, playing an important role. Other pumps and various control pathways are also implicated in tigecycline resistance. Overall, tigecycline is a potent therapeutic option for enterobacterial infections. Accurate detection of tigecycline susceptibility status and surveillance of resistant organisms in the hospital environment is necessary in order to optimise its use and to preserve tigecycline in our therapeutic arsenal.

In Vitro Activity of Tigecycline Against Acinetobacter baumannii: Global Epidemiology and Resistance Mechanisms.

Acinetobacter baumannii is a pathogen of increasing concern, commonly causing outbreaks in the hospital environment. Of particular concern, A. baumannii strains exhibiting resistance to carbapenems, which were previously considered the treatment of choice for infected patients, have dramatically increased worldwide, leaving a few antibacterial choices. Tigecycline, a broad-spectrum modified minocycline derivative, isconsidered as a last resort drug against multidrug-resistant A. baumannii. Though, resistance to tigecycline has emerged and is growing notably following increasing tigecycline usage. Comparative evaluation of the tigecycline resistance rates reported worldwide is challenging due to the absence of official interpretative criteria for in vitro susceptibility testing and the discrepancies among the different susceptibility methodologies used, with broth microdilution being considered the reference method. Tigecycline resistance is mainly associated with resistance-nodulation-cell division (RND)-type transporters, mainly the AdeABC, AdeFGH and AdeIJK efflux pumps, but other resistance mechanisms have also been implicated. Tigecycline is still an attractive choice for A. baumannii, but further investigations are warranted so that treatment of MDR Α. baumannii could be guided by validated in vitro data.

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates.

We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 µg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

The Role and Predictive Value of Cytokines in Atherosclerosis and Coronary Artery Disease.

Atherosclerosis is currently regarded as a chronic inflammatory disease that is mediated by several types of cells and molecules. Emphasis has been placed on the role of cytokines and the way they act and interact to initiate and sustain inflammation in the microenvironment of an atherosclerotic plaque. Cytokines are invariably expressed by all cells involved in the pathogenesis of atherosclerosis, act on a variety of targets exerting multiple effects and are largely responsible for the crosstalk among endothelial, smooth muscle cells, leukocytes and other vascular residing cells. In the present paper our aim is to review current information on the role of the most commonly discussed cytokines in the process of atherogenesis and to discuss the prognostic significance of these cytokines in atherosclerosis and coronary artery disease.

Circulating Biomarkers Determining Inflammation in Atherosclerosis Progression.

Atherosclerosis is the main underlying pathology of cardiovascular disease and is precipitated by various hereditary and non-hereditary risk factors. Inflammation is considered an important step in the progression of atherosclerosis and involves numerous cells, mediators and cellular procedures. Therefore, a biomarker able to determine the vascular inflammatory status is imperative as the combination of inflammatory biomarkers with the classic risk factors might provide further information about atherosclerosis progression and cardiovascular risk. The identification of novel inflammatory molecules and the improvement in analytical methods allows the potential implementation of these tests in every day clinical practice. In the current article, we focus on the role of established and novel biomarkers in atherosclerosis progression and in the determination of cardiovascular risk. We also present recent data concerning the risk stratification of patients according to their inflammatory status and the possible anti-inflammatory treatment strategies.

Is there any potential clinical impact of serum phosphorus and magnesium in patients with lung cancer at first diagnosis? A multi-institutional study.

BACKGROUND: The aim of the study was to determine whether the expression of baseline phosphorus (P) and magnesium (Mg) levels were prognostic in terms of stage and overall survival (OS) in newly diagnosed non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients. MATERIALS AND METHODS: Retrospectively, 130 patients were selected at the time of diagnosis oflung cancer (100 with NSCLC and 30 with SCLC), before the initialization of any chemo-radiotherapy. The median age was 67 (range 29-92). IA, IB, IIA, IIB, IIIA, IIIB and IV stages were present in 3, 4, 19, 6, 25, 8, and 65 patients, respectively. After centrifugation, the levels of serum P and Mg were measured using the nephelometric method/ photometry and evaluated before any type of treatment. RESULTS: Higher than normal levels of P were found in 127/130 patients, while only four patients had elevated Mg serum values. In terms of Spearman test, higher P serum values correlated with either stage (rho=- 0.334, p<0.001) or OS (rho=-0.212, p=0.016). Additionally, a significant negative correlation of Mg serum levels was found with stage of disease (rho=-0.135, P=0.042). On multivariate cox-regression survival analysis, only stage (p<0.01), performance status (p<0.01) and P serum (p=0.045) showed a significant prognostic value. CONCLUSIONS: Our study indicated that pre-treatment P serum levels in lung cancer patients are higher than the normal range. Moreover, P and Mg serum levels are predictive of stage of disease. Along with stage and performance status, the P serum levels had also a significant impact on survival. This information may be important for stratifying patients to specific treatment protocols or intensifying their therapies. However, larger series are now needed to confirm our results.

Cytomegalovirus reactivation in a general, nonimmunosuppressed intensive care unit population: incidence, risk factors, associations with organ dysfunction, and inflammatory biomarkers.

PURPOSE: Cytomegalovirus (CMV) reactivation, a significant cause of morbidity and mortality in immunosuppression, may affect "immunocompetent" seropositive critically ill patients. The aim of this prospective, observational study was to define the incidence, risk factors, and the association with morbidity and mortality of CMV reactivation in a general population of critically ill immunocompetent patients. We also studied the relationship between reactivation and patients' inflammatory response, as expressed by cytokine levels and stress up-regulation by salivary cortisol. METHODS: This study included mechanically ventilated CMV-seropositive patients. A quantitative real-time polymerase chain reaction (PCR) was performed for CMV plasma DNAemia determination, upon intensive care unit (ICU) admission and weekly thereafter until day 28. Cytomegalovirus reactivation was defined as CMV plasma DNAemia greater than or equal to 500 copies/mL. Upon ICU admission, interferon γ, interleukin (IL) 10, IL-17A, IL-2, IL-6, and tumor necrosis factor α were quantified in plasma, and morning saliva was obtained to measure cortisol. Disease severity was assessed by Acute Physiology and Chronic Health Evaluation II score, whereas the degree of organ dysfunction was quantified by Sequential Organ Failure Assessment score. Mortality, duration of mechanical ventilation, and ICU length of stay were recorded. RESULTS: During the study period, 80 (51 men) patients with a median age of 63 years fulfilled the inclusion criteria. Reactivation of CMV occurred in 11 patients (13.75%). Median day of reactivation was day 7 post ICU admission. Total number of red blood cell units transfused (odds ratio [OR], 1.50; confidence interval [CI], 1.06-2.13; P = .02) and C-reactive protein levels upon ICU admission (OR, 1.01; CI, 1.00-1.02; P = .02) were independently associated with CMV reactivation. High IL-10 was marginally related to reactivation (P = .06). Sequential Organ Failure Assessment scores were higher in the group with CMV reactivation compared with patients without reactivation during the entire 28-day observation period (P < .006). Salivary cortisol, mortality, length of ICU stay, and duration of mechanical ventilation were similar in the 2 groups. CONCLUSIONS: Cytomegalovirus reactivation occurred in 13.75% of critically ill, immunocompetent patients. The degree of inflammation and the total number of transfused red blood cells units constituted risk factors. Cytomegalovirus reactivation was associated with more severe of organ dysfunction, but not with a worse clinical outcome.