In vivo standardization of cutaneous bactericidal activity of antiseptics by using monoxenic hairless mice

This study was designed to investigate the bactericidal activities of antiseptics on the cutaneous flora of hairless mice monoxenic to Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa in vivo. A standardized method for testing such antiseptics to compare their bactericidal effectiveness in humans in described. Seven antiseptics belonging to seven different chemical groups (iodine derivatives, alcohols, mercury compounds, quaternary ammonium salts, biguanides, phenols, and carbanilides) were used as recommended by the manufacturers (conditions of contact or prolonged contact time followed by washing with distilled water). Germfree hairless mice were infected with various bacterial strains by gastric intubation, producing levels of about 10(3) CFU/cm2 of skin. The antiseptic under test was placed on the right or left side of the ventral region. The contralateral side served as a control. In order to standardize the method, a number of crucial parameters were carefully controlled: the amount of antiseptic applied, the area of skin treated, the duration of treatment, and the washing procedure. Skin samples were obtained by cutaneous biopsy, which effectively removed all the bacteria along with the sample. The bacterial populations were counted before and after application of the antiseptic. Reductions of between 0.5 and 1.9 log units were obtained; these are comparable to those observed in humans. The standardization of our procedure and the use of animals with a strictly controlled flora eliminated much of the variability and sources of error inherent in human studies. This model could be of value for the study of resistant bacterial strains responsible for nosocomial infections and for investigations of damaged skin.     

Clinical use of blood,blood components and blood products.

The goal of modern transfusion therapy is to provide appropriate replacement therapy with blood components as opposed to whole blood for patients with specific hematologic deficiencies. A prerequisite of component therapy is, therefore, correct identification of the deficiency. Appropriate use of components avoids many of the hazards associated with the use of whole blood, and at the same time makes maximal use of this valuable resource. Blood components separated from whole blood soon after collection and appropriately stored can, in combination, provide all the factors present in fresh whole blood. Red cell concentrates prepared from multiple packs have a hematocrit of approximately 70%. They may be stored for up to 3 weeks at 4 degrees C and are recommended for most situations requiring red cell transfusions. Platelet concentrates, which can be stored for up to 72 hours at 22 degrees C, may be used for thrombocytopenic patients. Fresh frozen plasma, stored plasma, cryoprecipitated factor VIII, factor VIII concentrate and factor IX complex concentrate are available for the proper treatment of patients with hemorrhagic disorders due to coagulation factor deficiencies. Similarly, albumin and immune serum globulin are available for their oncotic and antibody properties respectively. Thus, the availability and appropriate use of the various blood products allows not only optimal transfusion therapy for each patient, but also fuller utilization of national blood resources.     

In vivo standardization of cutaneous bactericidal activity of antiseptics by using monoxenic hairless mice.

This study was designed to investigate the bactericidal activities of antiseptics on the cutaneous flora of hairless mice monoxenic to Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa in vivo. A standardized method for testing such antiseptics to compare their bactericidal effectiveness in humans in described. Seven antiseptics belonging to seven different chemical groups (iodine derivatives, alcohols, mercury compounds, quaternary ammonium salts, biguanides, phenols, and carbanilides) were used as recommended by the manufacturers (conditions of contact or prolonged contact time followed by washing with distilled water). Germfree hairless mice were infected with various bacterial strains by gastric intubation, producing levels of about 10(3) CFU/cm2 of skin. The antiseptic under test was placed on the right or left side of the ventral region. The contralateral side served as a control. In order to standardize the method, a number of crucial parameters were carefully controlled: the amount of antiseptic applied, the area of skin treated, the duration of treatment, and the washing procedure. Skin samples were obtained by cutaneous biopsy, which effectively removed all the bacteria along with the sample. The bacterial populations were counted before and after application of the antiseptic. Reductions of between 0.5 and 1.9 log units were obtained; these are comparable to those observed in humans. The standardization of our procedure and the use of animals with a strictly controlled flora eliminated much of the variability and sources of error inherent in human studies. This model could be of value for the study of resistant bacterial strains responsible for nosocomial infections and for investigations of damaged skin.     

An evaluation of the suitability of the European suspension test to reflect in vitro activity of antiseptics against clinically significant organisms

The effectiveness of four antiseptics representing soluble phenolics (Dettol), Quaternary Ammonium Compounds (QAC) (Dettol Hospital Concentrate: DHC), mixed QAC/chlorhexidine (Hibicet Hospital Concentrate: HHC) and povidone iodine (Betadine) was assessed using the proposed phase 2 step 1 European Suspension test. The in vitro activity of the antiseptics against two of the proposed challenge strains, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, was compared with that of 14 problematic clinical isolates of bacteria from a range of genera, including some multiple antibiotic resistant strains, and a clinical isolate of Candida albicans. In addition to the 5 min contact time recommended in the European test, a 1 min time was included. All four products, at their recommended use dilutions and a contact time of 5 min, achieved a Microbicidal Effect (ME) log reduction of at least 5 against the majority of organisms. Differences in activity between products were more pronounced and therefore the tests more discriminatory, when the contact time was reduced to 1 min. The clinical strains were not overtly more resistant to antiseptics than the standard test strains, suggesting that the CEN test strains mimic the antiseptic susceptibility of clinical isolates.     

Antiseptic resistance of clinical strains of Staphylococcus aureus

To evaluate the activity of antiseptics and the sensitivity-resistance of bacteria to antiseptics, a number of characteristics has been used, including the minimum inhibiting concentration for different strains, the frequency of statistical and clinical resistance, the antiseptic activity index. A wide spread of S. aureus strains isolated from patients with hospital infections has been revealed. Differences in the resistance of bacterial strains have been established, depending on the type of the antiseptic and the ecovar of bacteria: among hospital ecovars, resistant strains occur more frequently and can resist a wider range of antibiotics. In staphylococcal hospital ecovars the occurrence and level of resistance to a number of widely used antiseptics increase with time. In connection with a wide spread of staphylococcal hospital strains resistant to antiseptics, measures on the control of the circulation of such strains should be introduced into hospitals, and the data thus obtained should be used for the periodic reevaluation of antiseptics used in medical practice and for the choice of preparations to be used for individual therapeutic and prophylactic antisepsis.     

Effect of blood storage on radiation-induced micronuclei in human lymphocytes.

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects (n=11), and from cancer patients (n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137Cs-irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22 degrees C) or in the refrigerator (5 degrees C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22 degrees C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5 degrees C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22 degrees C or at 5 degrees C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5 degrees C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.     

Efficacy of two ethanol-based skin antiseptics on the forehead at shorter application times

Background  

Recent research suggests that alcohol-based skin antiseptics exhibit their efficacy on the resident skin flora of the forehead in less than 10 minutes. That is why we have looked at the efficacy of two ethanol-based skin antiseptics applied for 10, 2.5 and 2 minutes on skin with a high density of sebaceous glands. Each experiment was performed in a reference-controlled cross-over design with at least 20 participants. Application of isopropanol (70%, v/v) for 10 minutes to the forehead served as the reference treatment. The clear (skin antiseptic A) and coloured preparations (skin antiseptic B) contain 85% ethanol (w/w). Pre-values and post-values (immediately after the application and after 30 min) were obtained by swabbing a marked area of 5 cm² for about 10 s. Swabs were vortexed in tryptic soy broth containing valid neutralizing agents. After serial dilution aliquots were spread on tryptic soy agar. Colonies were counted after incubation of plates at 36°C for 48 h. The mean log₁₀ reduction of bacteria was calculated. The Wilcoxon matched-pairs signed-ranks test was used for a comparison of treatments.     

新型杀菌剂在造纸生产流程中的中试研究

造纸白水中的可溶性有机物以及造纸系统的pH值、温度等环境条件都极其有利于微生物生长。当白水中的微生物含量大于10~8CFU/mL,造纸系统就会产生腐浆,进而引起断纸、洞眼等问题。因此造纸系统要使用杀菌剂抑制微生物生长。本研究采用两种新型杀菌剂分别进行了实验室和中试抑菌试验,结果表明在两种杀菌剂协同作用下,造纸系统没有明显的腐浆产生。     

Sample handling and stability of hepatocyte growth factor in blood samples

As regards clinical studies performed on hepatocyte growth factor (HGF) during recent years, we have aimed in the present study to investigate the eventual differences in sample handling of this cytokine that might influence the results of serum concentrations. Venous blood from patients with current infectious diseases and controls was used in different sub-studies. Compared with samples separated within one hour, no significant changes in serum HGF levels were observed when whole blood stayed 4, or 24h at 6 degrees C before or 6h in room temperature after separation but HGF levels were significantly higher (P<0.01) when whole blood was kept at room temperature 4 and 24h before separation. Serum HGF was stable up to 20 freeze-thaw cycles. The serum concentrations of HGF were significantly higher than levels in the plasma (19%; P<0.05). A significant increase in serum HGF levels (12%, P<0.05) was observed after shaking the whole blood sample to a visible haemolysis, although the HGF concentration in blood cells was around half of that in serum. HGF tolerated storage at -70 degrees C for at least 4 months. We conclude that standardized methods in sample handling are important in the study of HGF concentrations in blood samples.     

Use of whole blood directly for single-cell gel electrophoresis (comet) assay in vivo and white blood cells for in vitro assay

The present study investigated the use of whole blood from humans and rats directly for single-cell gel electrophoresis (comet) assay. As little as 20 microl of whole blood was sufficient for comet assay, and the comet images obtained from whole blood were not different from those obtained from isolated lymphocytes. The DNA remained intact up to 4 h at 4 degrees C after isolation and had no observable strand breakage, when whole blood was cryopreserved (at -80 degrees C) in 10% pre-cooled DMSO up to 60 days. To demonstrate that the whole-blood technique could be applied to in vivo studies, we injected rats with a known carcinogen Fe/NTA and measured DNA strand breaks in whole blood in comparison with isolated lymphocytes. We showed that Fe/NTA injection resulted in similar extent of DNA strand breakage in both whole blood and lymphocytes, indicating that whole-blood method can be used for in vivo genotoxic studies. One disadvantage of the whole-blood technique is that whole blood cannot be used for in vitro studies because of the interferences from red blood cell (RBC) components. However, this problem can be overcome by prior hemolysis of RBCs and a brief centrifugation to obtain white blood cells (WBCs), which can then be used for in vitro incubation with genotoxic compounds before comet assay. Overall, this whole-blood technique for comet assay is expected to provide a simple, rapid, and cost-effective alternative for the existing comet assay using isolated lymphocytes in situations such as when time and cost are limiting factors.     

An in vivo method for assessing the antimicrobial properties of topical antiseptics using artifically contaminated skin

A method is described for assessing the antimicrobial activity of topical antiseptics on human skin. The test, carried out on the forearms of volunteers, uses an inoculum of Stuphylococcus aureus . Up to four preparations may be tested at one time plus a placebo and untreated control site. The method has been used to compare four proprietary antiseptics and to evaluate their activity for up to one hour and has demonstrated a chloroxylenol, triclosan, EDTA combination product to be the most effective. The method is highly adaptable and may be used to evaluate activity versus other organisms and to examine other factors affecting antiseptic efficacy     

In vivo targeting of inflamed areas by electroloaded neutrophils.

Due to their spontaneous accumulation in inflamed or infected areas, blood phagocytes are potent drug vectors with specific targeting. Drug like molecule loading was obtained by use of cell electropermeabilization in which the impermeability of their plasma membrane is transiently impaired. Electrical conditions were used which allow electroloading of a drug like molecule (propidium iodide) in 70% of leukocytes in a whole blood sample while preserving in vitro functional properties. Slow release of entrapped hydrophilic molecules was observed with a half lifetime longer than 4 hours at 4 degrees C and at 37 degrees C. With an in vivo assay, using a rat model of inflammation, we showed that, as for non-pulsed cells, pulsed neutrophils accumulate 10 times more in an inflamed area than they do in control areas. Phagocyte electropermeabilization is therefore a very efficient way of drug targeting. Accumulation of electropulsed neutrophils in an area of inflammation gives targeted release of the electroloaded drug.     

Use of impedance methods for testing bactericidal activity of antiseptics

Standards PN-EN 1040 and EN 12054 describe test methods and minimum requirements for bactericidal activities of antiseptics. However, standard procedures are time consuming and require 48 hours of incubation. New alternative technique based on impedimetric procedure provides possibilities to reduce this time to several hours. The Bactometer (BioMerieux, Vitek System, USA) is a fully automated impedance technology system used to microbial quantitation of products. Impedance measures microbial activity by electrical methods. The aim of the study was to adaptate the impedimetric method utilising Bactometer--system to microbiological activity control of chemical antiseptics. Eight different products were utilised throughout the study. The samples for classical and alternative method were prepared in the same way as described in standards. The method of choice was dilution-neutralization method. All procedures conducted in Bactometer were verified by plate count method. The high correlation was observed between results obtained by normative methods and impedimetric measurement. All tested products meet requirements. The procedure utilising the Bactometer, provides a rapid and accurate system for the determination of bacterial content. The results of validation carried out during this study indicate, possibility to use impedimetric method alternatively to traditional methods.     

Comparison of surface plasmon resonance and quartz crystal microbalance in the study of whole blood and plasma coagulation

The coagulation of blood plasma and whole blood was studied with a surface plasmon resonance (SPR) based device and a quartz crystal microbalance instrument with energy dissipation detection (QCM-D). The SPR and QCM-D response signals were similar in shape but differing in time scales, reflecting differences in detection mechanisms. The QCM-D response time was longer than SPR, as a physical coupling of the sample to the substrate is required for molecules to be detected by the QCM-method. Change of sample properties within the evanescent field is sufficient for detection with SPR. Both the SPR signals and the QCM-D frequency and dissipation shifts showed dependency on concentrations of coagulation activator and sensitivity to heparin additions. The ratio of dissipation to frequency shifts, commonly considered to reflect viscoelastic properties of the sample, varied with the concentration of activator in blood plasma but not in whole blood. Additions of heparin to the thromboplastin activated whole blood sample, however, made the ratio variation reoccur. Implications of these observations for the understanding of the blood coagulation processes as well as the potential of the two methods in the clinic and in research are discussed.     

Comparison of rat blood preparation methods for acetaldehyde assay

A comparison is made of four previously described methods for the preparation of blood for acetaldehyde (AcH) assay in the rat. The spontaneous formation of AcH which occurs during the treatment of blood containing ethanol and the recovery of known amounts of AcH added to the blood were studied. The methods using sodium nitrite-sulfosalicylic acid or perchloric acid (PCA) in saline gave low levels of spontaneous formation (1 to 2 microM AcH for 48 mM ethanol). In the recovery studies it was seen that semicarbazide does not allow displacement of all the AcH; treatment of the blood with the reactant sodium nitrite-sulfosalycilic acid and use of the hemolysis method gave levels of recovery lower than 50%. Only treatment of the blood with perchloric acid in NaCl allowed all the AcH added to the blood to be recovered. In vivo, PCA in saline releases the AcH which was seen to remain bound in the red blood cells with the semicarbazide method. So the recommended procedure for accurate assay of blood AcH in the rat is cold deproteinization in PCA/saline before head-space gas chromatography. The levels of in vivo blood AcH (4.1 +/- 0.33 microM) obtained in the rat using this method for a blood alcohol concentration of 52 mM are lower than those previously described in the literature.     

In vivo protocol for testing efficacy of hand-washing agents against viruses and bacteria: experiments with rotavirus and Escherichia coli

Ten antiseptic formulations, an unmedicated liquid soap, and tap water alone were compared for their capacities to eliminate human rotavirus from the finger pads of adult volunteers; three of the antiseptics, the soap, and the tap water alone were also tested against Escherichia coli. A fecal suspension of virus or bacterium was placed on each finger pad and air dried. The contaminated site was exposed to the test product for 10 s, rinsed in tap water, and dried on a paper towel. The residual virus or bacterium was then eluted. Selected agents were also tested by an analogous whole-hand method by which the entire palm surfaces of both hands were contaminated. Alcohols (70%) alone or with Savlon reduced the virus titer by greater than 99%, whereas the reductions by Proviodine, Dettol, and Hibisol ranged from 95 to 97%. Aqueous solutions of chlorhexidine gluconate were significantly less effective for virus removal or inactivation than 70% alcohol solutions. Furthermore, Savlon in water (1:200) was found to be much less effective in eliminating the virus (80.6%) than the bacterium (98.9%). The tap water alone and the soap reduced the virus titers by 83.6 and 72.5% and the bacterial titers by 90 and 68.7%, respectively. The results of the whole-hand method agreed well with those of the finger pad protocol. We conclude that the finger pad method is a suitable model for testing the in vivo efficacy of hand-washing agents and emphasize the need for using appropriate test viruses and bacteria.     

Combined antibacterial effects of tissue‐tolerable plasma and a modern conventional liquid antiseptic on chronic wound treatment

Potential antimicrobial effects of sequential applications of tissue‐tolerable plasma (TTP) and the conventional liquid antiseptic octenidine dihydrochloride (ODC) were investigated. 34 patients with chronic leg ulcers were treated with TTP, ODC or a combination of both. The bacterial colonization was measured semi‐quantitatively before and immediately after treatment and changes in the microbial strains' compositions before and after antiseptic treatments were analyzed. All antiseptic procedures reduced the bacterial counts significantly. The sequential application of TTP and ODC displayed the highest antimicrobial efficacy. Me combined use of TTP and conventional antiseptics might represent the most efficient strategy for antiseptic treatment of chronic wounds. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

On the in vivo material functions of mammalian blood

The present paper explores the implications of employing time-averaged true continuum fields to investigate blood rheology in "steady viscometric" flows. This approach is in contrast with the spatially-averaged interpretation of field variables which is generally employed. On the basis of four plausible constitutive assumptions it is then possible to deduce the qualitative in vivo behavior of all three of the material functions of whole mammalian blood from inspection of the corresponding velocity profile. Quantitative results, and the evaluation of the material constants for specific constitutive models, can be obtained through curve-fitting procedures, as is illustrated. The development reconfirms, and puts on a formal basis, the earlier conclusion of Bugliarello, et al, that whole blood can have a dilatant response at low rates of shear. In addition, the normal stress forces are shown to have off-axis extrema in tube flow, and to be large enough to influence particle migration across streamlines. The existing data on particle migration in whole blood is reviewed, and shown to be in accord with these results.     

In Vitro Activity of Actinospectacin in Human Whole Blood

A method for testing antibacterial substances in whole blood is described. The test agent for the method was actinospectacin which reportedly has good in vivo activity, approximately in the range with chloramphenicol, but relatively poor in vitro activity in the common media. In human whole blood, however, the in vitro activity compares favorably with chloramphenicol thus indicating that whole blood may predict in vivo activity better than the usual bacteriological media.     

Cell type–specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.