Human rotavirus A detection: Comparison of enzymatic immunoassay and rapid chromatographic test with two quantitative RT-PCR assays.

OBJECTIVE: The aim of this study was to compare results of two commercially available kits used for routine detection of Rotavirus A in human stool samples with results of commercial quantitative reverse-transcription PCR (RT-qPCR) test and in-house RT-qPCR. MATERIAL AND METHODS: In total, 749 stool samples were screen-ed with the use of four different methods. The samples were collected from four diagnostic laboratories from March 2016 to June 2017. Diagnose of gastrointestinal disorders was stated in one third of tested patients, the rest of samples was collected from patients with other primary diagnose. The samples were tested with the enzymatic immunoassay (EIA) (RIDASCREEN® Rotavirus) and with rapid diagnostic immunochromatographic test (RDT) (IMMUNOQUICK® No-Rot-Adeno). As a reference method a commercial RT-qPCR test was used (Primerdesign Genesig® Kit) and it was compared with in-house RT-qPCR test prepared in our laboratory. The samples which in the reference RT-qPCR gave positive signal of reaction in cycle 28 or higher (Ct 28) were assessed as negatives in order to include only samples with some clinical relevance into sensitivity determination. RESULTS: Diagnostic sensitivity was assessed as 84.2% for EIA and 82.5% for RDT. The specificity of those tests was calculated as 97.8% for EIA and 96.4% for RDT. The performance of both diagnostic tests describing their positive predictive value was determined to be 87.3% for EIA and 80.3% for RDT. Negative predictive value was calculated to be 97.2% for EIA and 96.8% for RDT. Proportion of RVA-positive samples determined with the reference RT-qPCR test with our own cut-off level was 15.2% (n=114). Comparisons of the in-house and reference RT-qPCR tests showed very good agreement of results. The sensitivity of the in-house test was 100% and its specificity 99.7%. CONCLUSIONS: RT-qPCR is more sensitive for surveillance of rotavirus gastroenteritis than routinely used EIA or RDT methods. The specificity of both evaluated tests was very high. However, EIA was in all performance parameters assessed better than RDT.

ESCMID guideline for the diagnosis and treatment of biofilm infections 2014.

Biofilms cause chronic infections in tissues or by developing on the surfaces of medical devices. Biofilm infections persist despite both antibiotic therapy and the innate and adaptive defence mechanisms of the patient. Biofilm infections are characterized by persisting and progressive pathology due primarily to the inflammatory response surrounding the biofilm. For this reason, many biofilm infections may be difficult to diagnose and treat efficiently. It is the purpose of the guideline to bring the current knowledge of biofilm diagnosis and therapy to the attention of clinical microbiologists and infectious disease specialists. Selected hallmark biofilm infections in tissues (e.g. cystic fibrosis with chronic lung infection, patients with chronic wound infections) or associated with devices (e.g. orthopaedic alloplastic devices, endotracheal tubes, intravenous catheters, indwelling urinary catheters, tissue fillers) are the main focus of the guideline, but experience gained from the biofilm infections included in the guideline may inspire similar work in other biofilm infections. The clinical and laboratory parameters for diagnosing biofilm infections are outlined based on the patient's history, signs and symptoms, microscopic findings, culture-based or culture-independent diagnostic techniques and specific immune responses to identify microorganisms known to cause biofilm infections. First, recommendations are given for the collection of appropriate clinical samples, for reliable methods to specifically detect biofilms, for the evaluation of antibody responses to biofilms, for antibiotic susceptibility testing and for improvement of laboratory reports of biofilm findings in the clinical microbiology laboratory. Second, recommendations are given for the prevention and treatment of biofilm infections and for monitoring treatment effectiveness. Finally, suggestions for future research are given to improve diagnosis and treatment of biofilm infections.

Value of PCR in surgically treated patients with staphylococcal infective endocarditis: a 4-year retrospective study.

The aim of the study was to establish a diagnostic value for broad-range polymerase chain reaction (br-PCR) and staphylococci-specific multiplex PCR (ssm-PCR) performed on surgical material from patients with staphylococcal infective endocarditis (IE). Data were analysed retrospectively from 60 patients with suspected staphylococcal IE and 59 controls who were surgically treated at three cardiosurgery centres over 4 years. Both PCR tests showed high agreement and could be aggregated. In patients with definite and rejected IE, the clinical sensitivity and specificity of PCR reached 89 and 95%, respectively. Tissue culture (TC) and PCR agreed with blood culture (BC) in 29% and 67% of IE cases. TC helped to determine aetiology in five BC negative cases while PCR aided in nine cases. Out of 52 patients with conclusive staphylococcal IE, 40 were diagnosed with S. aureus and 12 with coagulase-negative staphylococci. PCR was shown to be highly superior to TC in confirming preoperative diagnosis of IE. In addition to aid in culture negative patients, PCR helped to establish or refine aetiology in inconclusive cases. We suggest that simultaneous br-PCR and ssm-PCR performed on surgical material together with histopathology could significantly increase the performance of current Duke criteria.

Bacterial growth on chitosan-coated polypropylene textile.

Biofouling is a problem common in all systems where microorganisms and aqueous environment meet. Prevention of biofouling is therefore important in many industrial processes. The aim of this study was to develop a method to evaluate the ability of material coating to inhibit biofilm formation. Chitosan-coated polypropylene nonwoven textile was prepared using dielectric barrier discharge plasma activation. Resistance of the textile to biofouling was then tested. First, the textile was submerged into a growth medium inoculated with green fluorescein protein labelled Pseudomonas aeruginosa. After overnight incubation at 33°C, the textile was observed using confocal laser scanning microscopy for bacterial enumeration and biofilm structure characterisation. In the second stage, the textile was used as a filter medium for prefiltered river water, and the pressure development on the in-flow side was measured to quantify the overall level of biofouling. In both cases, nontreated textile samples were used as a control. The results indicate that the chitosan coating exhibits antibacterial properties. The developed method is applicable for the evaluation of the ability to inhibit biofilm formation.

[Urinary catheter biofilm infections]. / Biofilmové infekce mocových katétru.

Urinary tract infections, most of which are biofilm infections in catheterized patients, account for more than 40% of hospital infections. Bacterial colonization of the urinary tract and catheters causes not only infection but also other complications such as catheter blockage by bacterial encrustation, urolithiasis and pyelonephritis. About 50% of long-term catheterized patients face urinary flow obstruction due to catheter encrustation, but no measure is currently available to prevent it. Encrustation has been known either to result from metabolic dysfunction or to be of microbial origin, with urease positive bacterial species implicated most often. Infectious calculi account for about 15-20% of all cases of urolithiasis and are often associated with biofilm colonization of a long-term indwelling urinary catheter or urethral stent. The use of closed catheter systems is helpful in reducing such problems; nevertheless, such a system only delays the inevitable, with infections emerging a little later. Various coatings intended to prevent the bacterial adhesion to the surface of catheters and implants and thus also the emergence of biofilm infections, unfortunately, do not inhibit the microbial adhesion completely and permanently and the only reliable method for biofilm eradication remains the removal of the foreign body from the patient.

[Detection of viable metabolically active yeast cells using a colorimetric assay]. / Prukaz zivotaschopných, metabolicky aktivních kvasinkových bunek pomocí kolorimetrického média.

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

Importance of biofilm in Candida parapsilosis and evaluation of its susceptibility to antifungal agents by colorimetric method.

The ability of C. parapsilosis (an important cause of nosocomial infections) to produce biofilm was evaluated in 32 bloodstream isolates and 85 strains isolated from skin. The biofilm formation was found in 19 (59%) blood isolates and only in 33 (39%) isolates from skin. The antifungal susceptibility was assessed for amphotericin B, itraconazole and voriconazole in planktonic and biofilm form of the 19 biofilm-positive bloodstream strains by broth microdilution method according to NCCLS standards. The method was modified by the use of resazurin as a colorimetric indicator of the metabolically active cells which makes the determination of the effect of antifungal agents easier. Biofilm forms of all strains were more resistant than their planktonic form.

Capillary isoelectric focusing of native and inactivated microorganisms.

The research of microorganisms includes the development of methods for the inactivation of viruses and other microbes. It also means to efficiently eliminate the infectivity of microorganisms without damage of their integrity and structure. According to the results of the last 5 years the capillary electromigration techniques appear to be very perspective for the comparison of the methods applicable for inactivation in the diagnostics and study of the pathogens. In this paper we suggest the capillary isoelectric focusing of the model microorganisms, Escherichia coli, Staphylococcus epidermidis, Candida albicans and bacteriophage PhiX 174, native or inactivated by different procedures. UV detection and fluorometric detection for the dynamically modified microbes by pyrenebutanoate on the basis of the non-ionogenic tenside were used here. Isoelectric points of native and/or dynamically modified microorganisms and other properties were compared with those obtained after microorganisms inactivation. The segmental injection of the sample pulse enabled the reproducible and efficient capillary isoelectric focusing in different pH gradients. The low-molecular-weight pI markers were used for tracing of the pH gradient.

[Detection and significance of biofilm formation in yeasts isolated from hemocultures]. / Detekce a význam tvorby biofilmu u kvasinek izolovaných z hemokultur.

BACKGROUND: Yeasts of the genus Candida are important opportunistic pathogens responsible for severe bloodstream infections in immunocompromised patients. An important virulence factor that allows them to colonize plastic implants and to survive in the blood is their ability to form biofilm. The purpose of the study was to assess, how frequently this virulence factor occurred in yeasts of the genus Candida isolated from blood cultures. MATERIALS AND METHODS: Cultures of the various strains in wells of microtitre plates were used to demonstrate the presence of biofilms. In total, we tested 72 isolates of yeasts of the genus Candida from the blood culture: C. albicans (33), C. parapsilosis (19), C. tropicalis (11), C. glabrata (4), C. krusei (3), C. lipolytica (1) and C. lusitaniae (1). RESULTS: The ability to form biofilm was demonstrated in 26 strains. The largest number of biofilm-positive strains was found in C. parapsilosis (13) and C. tropicalis (7). The formation of biofilms was far less frequent in the strains of C. albicans (4). The formation of biofilms was also demonstrated in C. krusei (2) and C. glabrata (1). CONCLUSION: The increasing frequency of fungal bloodstream infections is due to the increasing use of catheters and implants. The biofilm formation in these yeasts is the chief factor of virulence promoting such infections. The demonstration of biofilm formation in an isolated strain is indicative of the probable existence of a biofilm focus and of possible difficulties during antimycotic therapy.

[Methods for detection of biofilm formation in routine microbiological practice]. / Moznosti prukazu tvorby biofilmu v rutinní mikrobiologické praxi.

The increasing use of catheters, artificial implants and antimicrobials as well as high numbers of immunocompromised patients are major causes for concern over biofilm infections. These infections are characterized particularly by high resistance to antimicrobials and formation of persistent foci that may complicate therapy. Therefore, detection of biofilm formation is of high relevance to the clinician and his/her approach to the treatment. Reliable and sensitive methods for detection of this pathogenicity factor in clinically important organisms, suitable for use in routine microbiological laboratories, are needed for this purpose. Currently, a wide array of techniques are available for detection of this virulence factor, such as biofilm visualization by microscopy, culture detection, detection of particular components, detection of physical and chemical differences between biofilm-positive organisms and their planktonic forms and detection of genes responsible for biofilm formation. Since each of these methods has limitations, the best results can be achieved by combining different approaches.

Impact of surface coating on the adherence of slime producing and nonproducing Staphylococcus epidermidis.

The ability of Staphylococcus epidermidis to grow in the form of a biofilm not only facilitates its persistence in the host, but also allows it to survive at antibiotic concentrations several orders higher than the Minimum Inhibitory Concentration (MIC). We evaluated different surface treatments of hardened polystyrene in order to develop a model system for growth of S. epidermidis as a biofilm. We assayed for biofilm growth of S. epidermidis clinical isolates on unmodified polystyrene, on polystyrene modified by chemical abrasion and on polystyrene modified by sulfonation, using either Tryptic Soya Broth or Brain Heart Infusion as a growth medium. We concluded that sulfonated polystyrene and Brain Heart Infusion provided the best growth system for predicting the ability of a clinical isolate to form biofilm (Akaike value 23.680). Using this method, biofilm formation was detected in 14 (70%) of ica-positive strains and negative in 16 (80%) of ica-negative strains.

[Differences in antibiotic sensitivity in biofilm-positive and biofilm-negative strains of Staphylococcus epidermidis isolated from blood cultures]. / Rozdíly v citlivosti k antibiotikum u biofilmpozitivních a biofilmnegativních kmenu Staphylococcus epidermidis izolovaných z hemokultur.

The adhering capability and biofilm growth facilitate staphylococcal colonization of surfaces of damaged tissues and foreign bodies. Biofilm-forming bacteria are more resistant to immune system activities, mechanical effects of blood flow and other adverse effects, e.g. those due to antibiotics. Minimal inhibitory concentrations (MICs) were compared for two groups of Staphylococcus epidermidis strains isolated from blood cultures. Group 1 included biofilm positive strains whose biofilm-forming potential was revealed by both phenotypic and genotypic methods. Group 2 included strains without biofilm-forming potential. The comparison of MICs for selected antibiotics showed higher resistance of biofilm positive compared to biofilm negative strains. The difference was evident particularly for oxacillin, tetracycline, co-trimoxazole and gentamicin.

Biofilm detection and the clinical significance of Staphylococcus epidermidis isolates.

The ability of Staphylococcus epidermidis to produce biofilm was compared in 147 clinically significant strains repeatedly isolated from blood cultures of patients with bloodstream infection and in 147 strains isolated from skin. The strains were examined for the presence of ica operone, for the ability to form biofilm by Christensen's test-tube method and for the production of slime by Congo Red agar method. The ica operone was found in 92 (62.6 %) blood isolates and in 44 (29.9) isolates from skin. Christensen's test-tube method was positive in 79 (53.7) and 33 (22.4), Congo Red agar method in 64 (43.5) and 31 (21.1) of blood and skin isolates, respectively. All three methods were more frequently positive in clinically significant isolates from blood than in strains isolated from skin. The detection of ica operone and the Christensen's test-tube method showed better correlation with the clinical significance than the Congo Red agar method.

[In vitro evaluation of antibacterial activity of the original oxime type of beta-lactam antibiotics with the 4-aminobenzensulfonamide group]. / In vitro hodnocení antibakteriální úcinnosti puvodních beta-laktamových antibiotik oximového typu se 4-aminobenzensulfonamidoskupinou.

Four newly prepared beta-lactam antibiotics of the oxime type with a 4-aminobenzensulphonamidogroup, synthetized at the Research Institute for Pharmacy and Biochemistry, Prague, were subjected to an evaluation of antibacterial efficacy in vitro. The evaluation was carried out by determining the minimal inhibitory concentration (MIC) in 47 clinical strains and 6 standard strains (Staphylococcus aureus, Streptococcus pyogenes and facecalis, E. coli, Enterobacter and Proteus spec., Pseudomonas aeruginosa). The results were compared with the MIC of cefalexin, cefoxitin, cefazolin, cefsulodin, and ampicillin. The antibacterial effect of these agents was lower than that of the compared antibiotics. It was slightly better in the pseudomonadic strains, where the efficacy of cefusulodin was best and the other four antibiotics appeared to be worst.

[In vitro antibacterial activity of new oxime type of beta-lactam antibiotics]. / Antibakteriální úcinnost nových beta-laktamových antibiotik oximového typu in vitro.

The antibacterial efficacy of two novel beta-lactam antibiotics of the oxime type, VUFB 16265 and 16272, was evaluated by determining the minimal inhibitory concentration (MIC) in 47 clinical strains and 6 standard strains (Staphylococcus aureus, Streptococcus pyogenes and faecalis, E. coli, Enterobacter and Proteus spec., Pseudomonas aeruginosa), which were the most frequent causes of infectious complications. The results were compared with the MIC of cefalexin, cefoxitin, cefazolin, cefsulodin, and ampicillin. The antibacterial activity of both VUFB drugs was comparable with the efficacy of the antibiotics tested. Only in the strains of Pseudomonas aeruginosa the activity of all antibiotics under evaluation was low and only cefsulodin was effective.

[Determination of sulbactam in human serum using capillary isotachophoresis]. / Stanovení sulbaktamu v lidském séru kapilární izotachoforézou.

A method of determination of sulbaktam in human serum by capillary isotachophoresis with the use of a conductivity detector was worked out. Prior to the proper analysis, a pretreatment of the sample of serum was carried out by extracting sulbaktam to butyl acetate. The total yield of the proposed analytical procedure with an extraction first stage approaches 94%. The smallest determinable amount of the sample corresponds to 1 micrograms of sulbaktam in 1 ml of serum. The reported method was employed to evaluate the samples of sera of volunteers after intravenous administration of the dosage form sulbaktam-ampicillin (VUFB) and the foreign pharmaceutical preparation Unasyn (Pfizer). Statistically insignificant differences in pharmacokinetic parameters have confirmed that the preparations are highly bio-equivalent.

Control of Pseudomonas sepsis in mice with azlocillin combined with gentamicin.

The antibacterial activity of azlocillin VUFB and gentamicin was tested in vivo. To mice with Pseudomonas sepsis the test substances were injected either alone or combined, and the mortality was assessed. The test substances were freeze dried injectable azlocillin and gentamicin (Pharmachim). Azlocillin was injected into mice either intravenously or intramuscularly in graduated doses of 50-500 mg/kg once daily. The therapeutic effect was assessed on the basis of survival of the treated mice. Our expectation of a potentiated effect of the azlocillin-gentamicin combination was confirmed. After intramuscular administration of azlocillin plus gentamicin 90% of the mice survived, in comparison with 40% only after either azlocillin or gentamicin alone.

Pharmacokinetics of azlocillin after intramuscular application.

The first pharmacokinetic data are reported of azlocillin injected intramuscularly to adult probands. The blood serum and urinary levels of azlocillin were determined microbiologically in 8 healthy volunteers after intramuscular injection of 2 grams. Peak serum levels of azlocillin were found by the end of hour 1 after injection, average level was (55.06 +/- 10.25) mg/l. At hour 2 the average level was (33.64 +/- 6.46) mg/l. and at hour 4, (9.47 +/- 1.79) mg/l. The biological half-life was 1.4 hour. In urine, (41.2 +/- 7.7)% of the administered dose was eliminated within 8 hours on average.