Antibacterial efficacy of new commercially manufactured disinfectant substances against Salmonella typhimurium

The antibacterial effect of 19 new commercially manufactured disinfectant substances on a Salmonella typhimurium strain was studied. The substances tested represent 9 quaternary ammonium salts (QAT) and 11 combinated QAT with other ingredients. The antimicrobial efficacy was characterized by influencing the growth and reproduction of bacterial cells expressed either by MIC and ED₅₀ values (ED50 values represent concentration of substance in μ/ml which cause inhibition of growth by 50%), as well as by the inhibition of incorporation rate of [¹⁴C] adenine and [¹⁴C] leucine. The disinfectants are divided into three groups according to their efficacy. The first group comprises substances with strong inhibitory effect (MIC 0.04–0.19 μ/ml) such as Neoquat S, Antibacteric P, Divoquat forte, Sokrena and Diesin forte (sole from the group belonging to multicomponent substances). QAT except Antibacteric P interfere with energy metabolism (R values ～1). The second group represents substances with good antibacterial efficacy (MIC values up 1.56 μg/ml), and the third group substances with MIC values up 12.5 μg/ml. Cutasept G was found ineffective also in the concentration 100 μg/ml. The method of inhibition of [¹⁴C] precursors is suitable as one from possible criterion in evaluation of antibacterial efficacy of various synthetic substances.     

Persistent efficacy of topical eprinomectin against nematode parasites in cattle

Six studies were conducted to evaluate the persistent efficacy of eprinomectin pour-on against experimental challenges with infective nematode larvae in calves. In each study, calves were randomly assigned to one untreated group and up to four test groups, which were treated with eprinomectin at 500 μg/kg body weight at weekly intervals before single bolus challenge. The calves were necropsied approximately 4 weeks after challenge infection for nematode recovery. Eprinomectin pour-on provided ≥90% efficacy against challenge with Haemonchus placei, Trichostrongylus axei and T. colubriformis at 21 days after treatment and against Cooperia oncophora, C. punctata, C. surnabada, Dictyocaulus viviparus, Nematodirus helvetianus, Oesophagostomum radiatum and Ostertagia ostertagi at 28 days after treatment. Received: 14 April 2000 / Accepted: 18 May 2000     

量子点流式微球技术检测抗胰岛素抗体方法学建立

目的将量子点荧光探针应用于流式微球技术,创建出量子点流式微球技术检测人抗胰岛素抗体的方法并对其偶联率、精密度、线性范围、灵敏度进行初步评价。方法将胰岛素与羧基化微球偶联制备免疫诊断微球,与血清或标准品中人抗胰岛素抗体结合后,加入兔抗人IgG抗体,最后加入生物素化的羊抗兔IgG抗体和链霉亲和素化的量子点,使微球表面呈现荧光,并通过流式细胞仪检测平均荧光强度(mean fluorescent intensity,MFI)并记录。结果羧基化微球偶联胰岛素的性能显著强于空白微球,偶联0.5～3 h为最佳偶联反应时间,偶联成功的免疫诊断微球放置4℃冰箱至少可稳定50 d。微球偶联微球表面所携带荧光MFI与所测抗胰岛素抗体浓度成正比,量子点流式微球技术检测同一标本的灵敏度(3.74 pg/ml)、精密度(8.24%)、线性范围(3.74～5 000 pg/ml)都优于酶联免疫吸附试验所测得灵敏度(24.53 pg/ml)、精密度(12.31%)、线性范围(24.53～2 500 pg/ml)。结论本实验创建的量子点流式微球技术可用于人血清抗胰岛素抗体测定,其检测性能良好。     

A modified colorimetric assay of macrophage activation for intracellular cytotoxicity against Leishmania parasites

An in vitro method is described which colorimetrically assesses the activation of macrophages for intracellular cytotoxicity against the obligate intracellular parasite Leishmania donovani. The assay system uses a highly purified macrophage population derived from 10-day murine bone marrow cultures. These were infected in vitro as a suspension culture with viable L. donovani amastigotes and then exposed to activating agents. After 48 h the intracellular parasites were released by SDS lysis of the macrophages. Surviving Leishmania organisms were quantitated by their conversion of the chromophore MTT. The sensitivity of this method was comparable with the established method of [3H]dThd incorporation. This assay system has been used to show that there is a dual signal requirement (recombinant interferon-gamma and bacterial endotoxin (LPS] for effective activation of macrophages for leishmanicidal activity.     

Reevaluation of efficacy against nematode parasites and pharmacokinetics of topical eprinomectin in cattle

A study was conducted to confirm the efficacy of topical eprinomectin against nematodes and to evaluate the pharmacokinetics in cattle prevented from having physical contact with other cattle and from self-grooming. Sixteen male Brown Swiss calves were infected with larvae of recently isolated nematode parasites. Inoculation was scheduled so that the nematodes were expected to be adults at the time of treatment. Animals were blocked based on pretreatment body weight and randomly allocated to the untreated control group or the group treated with EPRINEX® Pour-On (Merial; 0.5 mg eprinomectin per kilogram body weight). Plasma samples were collected prior to and between 1 and 21 days following treatment and analysed for eprinomectin (B1a component) concentrations. For parasite recovery, identification and counting, animals were humanely euthanized 21 days after treatment. Calves treated with eprinomectin had significantly (p?99 % reduction) adult Dictyocaulus viviparus, Bunostomum phlebotomum, Cooperia oncophora, Cooperia surnabada, Cooperia punctata, Nematodirus helvetianus, Oesophagostomum radiatum, Ostertagia ostertagi, Ostertagia lyrata, and Trichostrongylus axei and inhibited fourth-stage Nematodirus and Ostertagia larvae than the controls. The main pharmacokinetic parameters of eprinomectin B1a were: AUC(inf), 124?±?24 day ng/mL; T _1/2, 5.2?±?0.9 days; and C _max, 9.7?±?2.2 ng/mL. Individual maximal concentrations were observed 3–7 days after treatment. This study confirmed the continued high level of efficacy of topically administered eprinomectin against a wide range of recently isolated nematodes. In addition, this study demonstrates that oral ingestion is not required to achieve adequate exposure for efficacy following topical administration of eprinomectin.     

多重耐药革兰阴性杆菌体外联合药效数据库初建

目的研究临床常用抗生素对多重耐药革兰阴性杆菌的协同和相加作用,初步建立体外联合药效数据库。方法选取磷霉素(PHOS)、左氧氟沙星(LEV)、头孢他啶(CAZ)、复方新诺明(SMZ)、哌拉西林/他唑巴坦(TZP)、头孢哌酮/舒巴坦(SCF)和亚胺培南(IMP)7种抗生素,两两组合为21种药物组。经超广谱β-内酰胺酶(ESBLs)和改良碳青霉烯灭活试验(mCIM)确认,172株多重耐药革兰阴性杆菌分为耐碳青霉烯肺炎克雷伯菌(20株,A组)、泛耐药的鲍曼不动杆菌(50株,B组)、产ESBLs的肠杆菌(62株,C组)和耐碳青霉烯的铜绿假单胞菌(40株,D组)4个耐药菌组。棋盘稀释法测定21种药物组对4个耐药菌组的体外联合药效,统计学分析采用Whonet5.6。结果172株待测菌株均对7种抗生素单药耐药。联合药效检测结果显示:4组均未出现拮抗作用;对A组耐药菌呈现协同作用的药物组有10组,部分协同作用的有14组,无相加作用,PHOS+LEV药物组协同百分率最高(30%,6/20);对B组耐药菌呈现协同作用的药物组有12组,部分协同作用的有10组,相加作用的有3组,LEV+SMZ药物组协同作用百分率最高(56%,28/50);对C组耐药菌呈现协同作用的药物组有14组,部分协同作用的有17组,相加作用的有16组,IMP+LEV药物组协同作用百分率最高(30.6%,19/62);对D组耐药菌呈现协同作用的药物组有12组,部分协同作用的有14组,相加作用的有13组,IMP+LEV药物组协同作用百分率最高(20%,8/40)。结论喹诺酮类、碳青霉烯类、磺胺类和磷霉素等药物联合应用,对多重耐药革兰阴性杆菌有较好的协同作用,治疗前行体外联合药效监测,可提高抗生素使用的准确性,临床应用价值较高。     

A novel binding assay to assess specificity of monoclonal antibodies

Dynabeads TALON are uniform superparamagnetic polystyrene beads of 1 microm diameter that bind 6-His-tagged recombinant proteins through Co++-affinity binding, and are normally used for protein purification. We have used these beads to bind 6-His-rNKp30 and 6-His-rNKp46 to use as a matrix for evaluating NKp30 and NKp46 mAb submitted to the 8th International Human Leukocyte Differentiation Antigen Workshop. We show that recombinant protein coated beads are an effective tool to evaluate the specificity and epitope reactivity of mAb.     

Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair

There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.     

Development of a quantitative flow cytometry-based assay to assess infection by Plasmodium falciparum sporozoites

The human malaria parasite Plasmodium falciparum causes the most deadly parasitic disease worldwide, necessitating the development of interventions that block infection. Yet, preclinical assays to measure inhibition of infection date from the 1980s and are based on microscopy. Here, we describe the development of a simple flow cytometric assay that can be used to quantitatively assess P. falciparum sporozoite infection in vitro in low and medium throughput. We demonstrate the utility of this assay for assessing both drug inhibition of infection and measuring efficacy of antibodies in blocking parasite infection. This methodology will aid in assessing functional antibody responses to vaccination and novel drugs that prevent mosquito-to-man transmission of malaria.     

Adaptation of different modifications of enzyme immunoassay and immune blot assay to assess the candidate vaccine HIVREPOL against HIV/AIDS

A preclinical trial of the vaccine HIVREPOL provided a complex of methods for assessing the identity and specific activity of vaccines against HIVIAIDS. The identity of "HIVREPOL" has been assessed by indirect enzyme immunoassay (EIA): the vaccine specifically binds the antibodies of the sera from HIV-infected individuals. Immune blot assay was the most informative method for assessing the identity of the candidate vaccine. The sera from HIVREPOL-vaccinated mice recognized the proteins gp41, p24, p55 of cultured HIV1 on "New-Lay-Blot1" strips. The bands corresponding to p24 were revealed in the line blots "Blot-HIV-1/2+O" and "INNO-LIA-HIV-Confirmation". The specific activity of the HIVREPOL vaccine was confirmed from the reactivity of sera of the mice vaccinated with recombinant proteins of the immunosorbents available in EIA test systems for the detection of HIV antibodies. Competitive EIA established the antigen-binding activity of sera from HIVREPOL-vaccinated mice against the native reference HIV-1 antigen.     

A disc-based quantitative carrier test method to assess the virucidal activity of chemical germicides

Suspension tests for virucidal activity of chemical germicides are easier to perform, but they normally do not present the test product with a strong enough challenge. In contrast, carrier tests, where the test virus is dried on an animate or inanimate surface, offer the test formulation a higher level of challenge because it first has to penetrate successfully the inoculum to gain access to and inactivate the target organism on the carrier. Since pathogens in nature are normally found adsorbed to surfaces and/or embedded in organic or cellular debris, the results of carrier tests are more relevant to predicting the activity of chemical germicides under field situations. The method described below uses discs (1 cm in diameter) of brushed stainless steel discs as carriers. Ten micro l of the test virus in a soil load is placed on each disc and the inoculum dried under ambient conditions. The dried inoculum is then exposed to 50 micro l of the test formulation or a control solution for a defined contact time at the specified temperature. EBSS (0.95 ml) is added to each carrier holder to dilute/neutralize the germicide, the inoculum eluted and the eluates titrated in cell cultures to determine the degree of loss in virus viability. At least five test and three control carriers are used in each test. Controls are also included to test for toxicity of the test formulation to the host cells and any interference sub-cytotoxic levels of the formulation may have on the ability of the virus to infect the cells. The method has been used with several types of human and animal pathogenic viruses to test the activity of all major classes of chemical germicides against them.     

Comparative study of the standard fluorescent antibody to membrane antigen (FAMA) assay and a flow cytometry-adapted FAMA assay to assess immunity to varicella-zoster virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.     

The use of ANAM to assess the side-effect profiles and efficacy of medication

Cognition has become increasingly important as an outcome measure in studies of medication. Traditional neuropsychological assessment is limited in its ability to detect subtle, medication-related changes and it is not suitable for the rapid serial assessment required in most clinical trials. Thus, investigators have turned to computerized neuropsychological assessment for its repeatability, sensitivity to subtle cognitive changes, and ease of administration. The automated neuropsychological assessment metrics (ANAM) is one such computerized battery that has been used to measure the effects of numerous CNS-active drugs. This paper is an exhaustive review of studies that have used ANAM to measure cognitive changes associated with pharmacological treatments. The benefits and limitations of using ANAM in clinical trials are discussed.     

Evaluation of the capacity of the SCGE assay to assess the genotoxicity of biomaterials

The comet test or SCGE assay, which is already widely used in other areas, has never been used to evaluate the mutagenic potential of medical biomaterials in the final form. The purpose of our study was thus to assess the comet test as a means of assessing the genotoxic potential of finished medical biomaterials. We used silicone elastomers with increasing concentrations of 4-nitroquinoline oxide, a genotoxic agent. Hydrogen peroxide was used as the positive control, and tissue culture polystyrene as the negative control. In our study, the comet test did not detect a significant difference in genotoxicity between the pure elastomer and the same elastomer containing 0.01 mg/ml 4-nitroquinoline oxide, but did detect a significant difference between two elastomers containing 0.01 and 0.3 mg/ml of 4-nitroquinoline oxide, respectively. Since, the surface properties of the samples were identical, only the chemical composition may have caused significant differences in mutagenicity. Whatever the cause of the genotoxicity detected by the SCGE assay, testing finished biomaterials using the comet assay makes it possible to evaluate interactions between biomaterials and living tissues that are much closer to actual application conditions.     

Kinetics of bactericidal and sporicidal effects of a disinfectant against bacteria isolated from hospital units]

The bactericidal and sporicidal activities of a peracetic acid disinfectant commonly used in hospitals (Acetoper 200) was studied using three bacterial species recovered in water from hemodialysis machines and humidifiers, i.e., Pseudomonas aeruginosa, Serratia liquefaciens, and Bacillus subtilis (spores). The method used was derived from AFNOR norms NF T 72-151 and NF T 72-231. For each strain, three concentrations of disinfectant were tested. Counts were performed every five minutes for one hour to evaluate killing kinetics. For both Gram-negative organisms, survival curves were biphasic, whereas B. subtilis counts decreased logarithmically. Both concentration and time influenced the 5 log10 decrease in counts. With S. liquefaciens and B. subtilis (spores), the 5 log10 decrease was not reached with low levels of disinfectant. In every case, the D value decreased substantially with increasing levels of disinfectant.