Droplet digital PCR as a novel dia-gnostic tool.

BACKGROUND: Nowadays, modern treatment methods for cancer patients are based on targeting specific molecules involved in cellular signaling system associated with tumor initiation and progression. The success of such approach depends on a correctly chosen dia-gnostic test with high sensitivity that identifies the occurrence and level of bio-markers in patients to select those who will respond and benefit from the treatment. The development of new technologies and the upgrades of the known ones contribute to the innovations in molecular characterization of cancer, which allows the detection of patients mutational status with high sensitivity and specificity. PURPOSE: Here, we discuss the utilization of the third-generation type of polymerase chain reaction (PCR), droplet digital PCR (ddPCR), in the molecular dia-gnostics of oncology diseases. According to the studies reported in our review, ddPCR represents a promising tool in genetic profiling of cancer patients. Therefore, the optimization and precise validation may enable gradual implementation of ddPCR into clinical practice in the field of oncology.

Results from the Survey of Antibiotic Resistance (SOAR) 2014-16 in Ukraine and the Slovak Republic.

Objectives: To determine antibiotic susceptibility in isolates of Streptococcus pneumoniae and Haemophilus influenzae collected in 2014-16 from Ukraine and the Slovak Republic. Methods: MICs were determined by CLSI broth microdilution and susceptibility was assessed using CLSI, EUCAST and pharmacokinetic/pharmacodynamic (PK/PD) breakpoints. Results: S. pneumoniae isolates collected in Ukraine (n = 100) showed susceptibility rates ≥97% for amoxicillin, amoxicillin/clavulanic acid, penicillin [intravenous (iv) non-meningitis] and fluoroquinolones, between 83% and 86% for oral penicillin, macrolides and cefaclor, and 75% for trimethoprim/sulfamethoxazole. Susceptibility was substantially lower in the Slovak Republic (n = 95). All isolates were susceptible to the fluoroquinolones, but susceptibility to penicillin, amoxicillin, amoxicillin/clavulanic acid, cefuroxime and trimethoprim/sulfamethoxazole varied between 61% and 64%, with only 44% of isolates susceptible to the macrolides. Susceptibility of H. influenzae was more homogeneous, with susceptibility to amoxicillin/clavulanic acid, ceftriaxone, cefuroxime, azithromycin and the fluoroquinolones seen in >90% of isolates by CLSI criteria in both countries. Much greater variability was seen across breakpoints, especially for azithromycin, cefaclor and cefuroxime. The ß-lactamase rate was 5.1% (5/98) in the Slovak Republic and 7.3% (7/96) in Ukraine, but the Slovak Republic also had a relatively high rate of ß-lactamase-negative-ampicillin-resistant (BLNAR) isolates (7.1%; 7/98). Conclusions: The variability found across these two neighbouring countries illustrates the need to monitor and publish national and local resistance patterns. This information is not only critical for effective empirical therapy but can also be used to help shape and support antimicrobial stewardship efforts in order to limit antibiotic resistance.

Impact of interleukin 13 (IL13) genetic polymorphism Arg130Gln on total serum immunoglobulin (IgE) levels and interferon (IFN)-γ gene expression.

This cross-sectional study was designed to investigate the extent of genetic susceptibility by targeting variants in interleukin (IL)-4/IL-13 signalling pathways leading to atopic disease in early childhood. We evaluated involvement of five single nucleotide polymorphisms IL4 C-590T, IL13 C-1055T, IL13 Arg130Gln, IL4RA Ile50Val and IL4RA Gln576Arg, in the control of serum total and antigen-specific immunoglobulin (Ig)E levels. Furthermore, we analysed their association with changes in gene expression of five cytokines having key roles in inflammatory and anti-inflammatory immune response [IL-4, IL-13, interferon (IFN)-γ, IL-8 and IL-10]. Total and antigen-specific IgE levels in serum and gene expression of selected cytokines in peripheral blood were measured in 386 children aged 1-8 years. TaqMan allelic discrimination, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and restriction fragment length polymorphisms (RFLP) methods validated by sequencing were used for genotyping. All genotypes for children with total and antigen-specific IgE levels in the normal range were in Hardy-Weinberg equilibrium. Gene expression analyses were carried out using TaqMan gene expression assays. We found elevated total IgE levels in carriers of IL13 Arg130Gln variant allele [odds ratio (OR) = 1·84; 95% confidence interval (CI) = 1·16-2·93]. This effect was more apparent for boys (OR = 2·31; 95% CI = 1·25-4·28). However, no significant association was observed for the other four variants examined. We found up-regulation of IFN-γ in children with elevated serum total IgE levels carrying the Arg130 allele (P = 0·005). No differences were found for IL4, IL8 or IL10, while IL13 gene expression was under the detection limit. IL13 Arg130Gln genotypes can play a role in genetic susceptibility to allergy via regulation of serum total IgE levels and affecting IFN-γ gene expression.

The emergence of Clostridium difficile PCR-ribotype 001 in Slovakia.

PURPOSE: The purpose of this study was to determine the incidence of Clostridium difficile infections (CDI) and to characterise the isolates in 14 departments of ten academic hospitals in Slovakia. METHODS: During a one-month study (September 2012) all unformed stool samples were investigated using a rapid test to detect the presence of GDH and toxins A/B. Positive samples were cultured anaerobically and C. difficile isolates were characterised by ribotyping, multiple-locus variable-number tandem repeats analysis, and gyrA, rpoB and ermB investigation. RESULTS: A total of 194 unformed stool samples were investigated and 38 (19.6 %) had a positive rapid test. Of 38 samples, 27 revealed a positive result for GDH and free toxins A/B in the stool, and 11 samples only for the presence of GDH. The mean CDI incidence in 2012 was 5.2 cases per 10,000 patient bed-days. Twenty C. difficile isolates were available for molecular analysis; seventeen belonged to PCR-ribotype 001 (85 %) whereas the remaining three isolates were identified as PCR-ribotypes 017, 078 and 449. MLVA of the PCR-ribotype 001 isolates identified two clonal complexes and a close genetic relatedness between isolates from six different hospitals. Molecular analysis of antibiotic-resistance determinants revealed the presence of ermB gene encoding resistance to the MLSB group of antibiotics in 90 % of isolates, and Thr82Ile amino acid substitution in the gyrA gene associated with resistance to fluoroquinolones in 85 % of isolates. CONCLUSIONS: We conclude that C. difficile PCR-ribotype 001 is the predominant PCR-ribotype in Slovakia with a strong potential for clonal spread and development of multidrug resistance.

Titanium dioxide nanoparticles: some aspects of toxicity/focus on the development.

Nanosized titanium dioxide (TiO2) particles belong to the most widely manufactured nanoparticles (NPs) on a global scale because of their photocatalytic properties and the related surface effects. TiO2 NPs are in the top five NPs used in consumer products. Ultrafine TiO2 is widely used in the number of applications, including white pigment in paint, ceramics, food additive, food packaging material, sunscreens, cosmetic creams, and, component of surgical implants. Data evidencing rapid distribution, slow or ineffective elimination, and potential long-time tissue accumulation are especially important for the human risk assessment of ultrafine TiO2 and represent new challenges to more responsibly investigate potential adverse effects by the action of TiO2 NPs considering their ubiquitous exposure in various doses. Transport of ultrafine TiO2 particles in systemic circulation and further transition through barriers, especially the placental and blood-brain ones, are well documented. Therefore, from the developmental point of view, there is a raising concern in the exposure to TiO2 NPs during critical windows, in the pregnancy or the lactation period, and the fact that human mothers, women and men in fertile age and last but not least children may be exposed to high cumulative doses. In this review, toxicokinetics and particularly toxicity of TiO2 NPs in relation to the developing processes, oriented mainly on the development of the central nervous system, are discussed Keywords: nanoparticles, nanotoxicity, nanomaterials, titanium dioxide, reproductive toxicity, developmental toxicity, blood brain barrier, placental barrier.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

The relationship between cell surface markers, cytokines, ageing, and cigarette smoking.

The purpose of this study was to investigate the modulation of selected cell surface markers and proinflammatory cytokines production in relation to ageing, and cigarette smoking. The analysis of cell surface receptors was performed by the flow cytometry and cytokines levels were evaluated by the sandwich enzyme immunoassays. We found a decreased expression of CD69, CD28, CD11b, CD95 markers in old population compared to young people (p<0.05; p<0.001). The memory CD45RO lymphocytes were markedly expanded in older population in comparison to young donors (12.93+/-5.92 %, p<0.001) and the selectin CD62L was significantly increased on granulocytes in aged people (p<0.05). Our findings demonstrated an augmented level of CD3 and CD28 on lymphocytes in smokers (p<0.05; p<0.005). The significant depression of CD16+56 molecule was recorded in smokers (10.86+/-0.80%) when compared to non-smokers (14.44+/-0.46; p<0.05). Our results showed a significantly diminished levels of interleukin (IL)-1beta (1.93+/-0.48 pg/ml), and increased levels of IL-6 and tumor necrosis factor (TNF)-alpha in elderly population compared to young people (p<0.05; p<0.001). The present data support previous suggestions that senescence and cigarette smoking may contribute to changes in the immune system activity, resulting in altered cell surface marker expression and cytokine levels (Tab. 1, Fig. 3, Ref. 81). Full Text (Free, PDF) www.bmj.sk.

Predictors of inferior outcome in community acquired bacterial meningitis.

The aim of this study was to assess mortality and sequellae within cases from Nationwide survey of community acquired meningitis and identify risk factors for inferior outcome. Risk factors such as underlying disease (diabetes mellitus, cancer, trauma, neonatal age, splenectomy, alcoholism, sepsis, other infections), etiology, clinical symptoms and outcome (death, improvement and cured after modifications of ATB therapy, cured without change of therapy, cured with neurologic sequellae) were recorded and analysed with univariate analysis (chi2 or t test for trends, CDC Atlanta 2004). Analysing risk factors for inferior outcome (death or cured with neurologic sequellae), we compared patients who died or survived with neurologic sequellae to all patients with community acquired bacterial meningitis. Univariate analysis showed that trauma (p<0.05), alcohol abuse (p<0.05), diabetes, S. aureus (p<0.05) and gram-negative etiology (A. baumannii, Ps. aeruginosa or Enterobacteriaceae) (36% vs. 11,9%, p<0.05) were predicting inferior outcome. Analysing risk factors for treatment failure (death or failed but cured after change of antibiotic treatment) prior sepsis (34.1% vs. 13.9%, p<0.01) and gram-negative etiology (25% vs. 11.9%, p<0.02) were statistically significant predictors of treatment failure. Neisseria meningitis had less failures (p<0.05). Concerning infection associated mortality again diabetes mellitus (p<0.05), alcoholism (p<0.05) staphylococcal and gram-negative etiology (p<0.05) were significant predictors of death. N. meningitis had surprisingly less treatment failures (appropriate and rapid initial therapy). Neurologic sequellae were more common in patients with alcohol abuse (p<0.05), craniocerbral trauma (p<0.05) and less common in meningitis with pneumococcal etiology (p<0.05).

Antimicrobial susceptibility of cerebrospinal isolates from patients with meningitis.

Str. pneumoniae isolates were susceptible to penicillin, all to also ofloxacin and chloramphenicol and cefotaxim and 39 (100%) to cotrimoxazol. Concerning S. aureus, all isolates 22 were susceptible to oxacillin and chloramphenicol, and 21 also to cotrimoxazol. All N. meningitidis isolates but one-10 of all were susceptible to penicillin, all to cefotaxim, chloramphenicol and cotrimoxazol. All H.influenzae isolates were susceptible to ampicillin and chloramphenicol, as well as to ofloxacin and cotrimoxazol. Those surprisingly high susceptibilities to rather "old" antibiotics may be explained by low antibiotic consumption, accessibility and therefore low usage which is a key promoter of resistance both in community and hospital.

Bacterial meningitis among Roma ethnic minority.

Etiology and risk factors such as malnutrition, diabetes, otitis/sinusitis, alcohol abuse, tuberculosis, low birth weigh as well as mortality and neurologic sequellea in Roma ethnic minority with community acquired bacterial meningitis (CBM) was assessed and compared to all CBM cases.

Etiology and resistance patterns of respiratory isolates in Kenyan adults with AIDS from slum population.

We investigated regularly swabs of adults dispenzarised at Mary Immaculate Clinic of Trnava University in Nairobi providing free health care for about 50 000 population of Mukuru Slums. 20 patients who were treated for AIDS by our clinic (those who started HAART before Free National AIDS Cooperation Programme - NASCOP) were assessed after 1, 2 and 3 years (18 of 20 completed the survey, other 2 loss of follow up, probably died. Exposure to other molecules can select resistant mutants. Previous exposure to TMP/SMX was similar in both groups and therefore was not responsible for the difference between resistance patterns.

Neuroinfections complicating foreign body implants after perinatal trauma or meningitis in 60 children.

Meningitis after artificial implants in 60 children, mainly after foreign body infections (FBI) was caused more frequently by coagulase negative staphylococci and Ps. aeruginosa than other organisms and was significantly associated with perinatal trauma, hydrocephalus, haemorrhage or VLBW and had more neurologic sequels despite mortality was similar to other nosocomial meningitis.

Four years of monitoring antibiotic resistance in microorganisms from bacteremic patients.

From the second semester of 2002 to the end of the first semester of 2005, a total of 2544 bacterial strains were isolated from the blood stream of patients with clinical sepsis and bacteremia hospitalized in six University Hospitals in the Slovak Republic. Almost 30% of strains were coagulase-negative staphylococci (CONS), about 14% were Staphylococcus aureus and, of the Gram-negative bacteria, up to 9% were Klebsiella pneumoniae. All CONS, S. aureus and Enterococcus spp. strains were found to be still susceptible to vancomycin, but the resistance of CONS and/or S. aureus to macrolides and fluoroquinolones dramatically increased during the period of this study. Among Gram-negative bacteria, increasing levels of resistance to higher generation cephalosporins, to fluoroquinolones resistance in Pseudomonas aeruginosa and Acinetobacter spp. to meropenem was recorded, which is alarming. The results were periodically submitted to cooperating hospitals with proposals for rationalizing the prophylactic and general use of indicated antibiotics as well as for improving hospital hygiene measures and anti-epidemic practices.

Occurrence of aminoglycoside-modifying-enzyme genes aac(6')-aph(2"), aph(3'), ant(4') and ant(6) in clinical isolates of Enterococcus faecalis resistant to high-level of gentamicin and amikacin.

The genes coding for 4 aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3'), ANT(4') and ANT(6) were determined in 44 Slovak clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin (HLGR, collection 1) and 48 E. faecalis isolates with resistance to amikacin (AR, collection 2). The occurrence of spotted genes was (collection 1 vs. collection 2): aac(6)-aph(2") 81.8 vs. 8.3 %, ant(4') 52.3 vs. 81.3 %, aph(3') 50 vs. 56.3 % and ant(6) 6.8 vs. 4.2 %, the most frequent combinations of genes in the HLGR collection were aac(6')-aph(2") + ant(4') and aac(6')-aph(2") + aph(3). In contrast, the aph(3') + ant(4') gene profile was predominant in AR isolates. None of the isolates contained all four AGME genes simultaneously.

[Potentiometric determination of aqueous dissociation constants of 2-,3-,4-{3-(4-benzhydryl-piperazine-1-yl)-2-hydroxy-propoxy}-phenylcarbamic acid alkylesters, water insoluble potential blockers of beta-adrenergic receptors]. / Potenciometrické stanovenie disociacných konstánt alkylesterov kyseliny 2-, 3-, 4-{2-hydroxy-3- -[(4-difenylmetyl)piperazin-1- -yl]propoxy}fenylkarbámovej, potenciálnych blokátorov beta-adrenergných receptorov nerozpustných vo vode.

Apparent ionization constants (W(s)pK(a)) of potential drugs, a series of twelve protonated bases, 2-,3-,4-{3-(4-benzhydryl-piperazine-1-yl)-2-hydroxy-propoxy}-phenylcarbamic acid alkylesters dihydrochlorides, were determined by automated potentiometric titrations in mixtures water - methanol. Aqueous pK were assessed by means of Yasuda-Shedlovsky equation. Approach for pKa determination was evaluated using trimecaine hydrochloride as the model compound. The first values of ionization constants ranged approximately from 2.7 to 3.2, the second ones from 6.5 to 7.2. Only a slight decrease in ionization constants with the number of carbon atoms was observed in homological series. Experimentally determined dissociation constants were compared with the values predicted by the computer program SPARC.

Bacteremia due to Pseudomonas aeruginosa: results from a 3-year national study in the Slovak Republic.

Risk factors, mortality and antimicrobial susceptibility of Pseudomonas aeruginosa bacteremias isolated from 148 patients from all University Hospitals in Slovakia were analyzed. Only 1.2% of 169 strains of P. aeruginosa were resistant to meropenem, 4.1% to piperacillin/tazobactam, 7.7% to ceftazidime as well as cefepime and 12% to amikacin. More than 30% of P. aeruginosa were resistant to ciprofloxacin. Our analysis of risk factors for antimicrobial resistance to the particular antimicrobials, indicated no difference in risk factors and outcome in cases infected with P. aeruginosa bacteremias resistant to amikacin, piperacillin/tazobactam or ceftazidime in comparison to episodes caused by P. aeruginosa due to susceptible isolates. When comparing risk factors for P. aeruginosa bacteremia in children vs. adults, cancer vs. non-cancer patients, several differences in risk factors were observed. Neither antimicrobial resistance to amikacin, ceftazidime or piperacillin/tazobactam, nor appropriateness of therapy according to two separate analyses were associated with better outcome.

DNA repair and cyclin D1 polymorphisms and styrene-induced genotoxicity and immunotoxicity.

1-SO-adenine DNA adducts, DNA single-strand breaks (SBs), chromosomal aberrations (CAs), mutant frequency (MF) at the HPRT gene, and immune parameters (hematological and of humoral immunity) were studied in styrene-exposed human subjects and controls. Results were correlated with genetic polymorphisms in DNA repair genes (XPD, exon 23, XPG, exon 15, XPC, exon 15, XRCC1, exon 10, XRCC3, exon 7) and cell cycle gene cyclin D1. Results for biomarkers of genotoxicity after stratification for the different DNA repair genetic polymorphisms showed that the polymorphism in exon 23 of the XPD gene modulates levels of chromosomal and DNA damage, HPRT MF, and moderately affects DNA adduct levels. The highest levels of biomarkers were associated with the wild-type homozygous AA genotype. The exposed individuals with the wild-type GG genotype for XRCC1 gene exhibited the lowest CA frequencies, compared to those with an A allele (P < 0.05). Cyclin D1 polymorphism seems to modulate the number of leukocytes and lymphocytes in the analyzed subjects. The number of eosinophiles was positively associated with XPD variant C allele and negatively with XRCC1 variant A allele (P < 0.05) and XPC variant C allele (P < 0.05). Immunoglobulin IgA was positively associated with an XRCC3 variant T allele (P < 0.01) and negatively with XPC variant C allele (P < 0.05). Both C3- and C4-complement components were lower in individuals with XRCC3 CT (P < 0.05) and TT genotypes (P < 0.01). Adhesion molecules sL-selectin and sICAM-1 were associated with XPC genotype (P < 0.05). Individual susceptibility may be reflected in genotoxic and immunotoxic responses to environmental and occupational exposures to xenobiotics.