Comparative testing of disinfectants using proposed European surface test methods

A surface test method for disinfectants currently under development as a standard European test method is described. The test involves determining the number of viable organisms recoverable from a contaminated stainless steel surface before and after application of disinfectant. Evaluation of a range of disinfectant agents and products currently used in the UK indicated that products at recommended use concentrations produced log reductions in viable count ranging from < 1.0 to > 6.4 (i.e. no detectable survivors) after a 5 min contact period against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium and Candida albicans. Increased activity was observed by increasing the disinfectant contact time to 30 min. Addition of 3% w/v albumin to the test suspension used to inoculate surfaces caused a substantial reduction in activity.     

European disinfectant testing — Collaborative trials

Collaborative trials were conducted on a basic bactericidal suspension test. This test was developed by the European Committee for the standardization of test methods (CEN TC 216). Two test materials were used, Phenol (A.R.) and Dodigen 2183. Interlaboratory agreement was very good, but it proved difficult to obtain precision data. Work is continuing.     

Bioluminescence for USP sterility testing of pharmaceutical suspension products

Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability of the current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilution step required for testing suspension products. Thirteen strains of bacteria and fungi (representing potential contaminants in sterile products), three pharmaceutical suspension products, and four media were used in the experiment. No interference from suspension products was encountered in the detection of microbial growth by the bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusion of oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexing the medium once during the 2-week incubation period. Bioluminescence measurement would facilitate automated handling of the sterility test endpoint readout operation. The optimum parameters of bioluminescence measurement for application in sterility testing were determined.     

Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M(r) value of 57,190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27-61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein is synthesized as a 557-amino acid precursor and processed to produce a mature protein of M(r) 54,505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.     

Bioluminescence for USP sterility testing of pharmaceutical suspension products.

Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability of the current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilution step required for testing suspension products. Thirteen strains of bacteria and fungi (representing potential contaminants in sterile products), three pharmaceutical suspension products, and four media were used in the experiment. No interference from suspension products was encountered in the detection of microbial growth by the bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusion of oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexing the medium once during the 2-week incubation period. Bioluminescence measurement would facilitate automated handling of the sterility test endpoint readout operation. The optimum parameters of bioluminescence measurement for application in sterility testing were determined.     

CEN/TC 216: its role in producing current and future European disinfectant testing standards

CEN, the European Committee for Standardization, is a legal association comprising of National Standards Bodies (e.g. AFNOR, BSI, DIN) responsible for the production of European Standards (ENs) which are designed to facilitate the exchange of goods and services through the elimination of technical barriers to trade. CEN/TC 216 was conceived in the late 1980s with the scope Standardization of the terminology, requirements, test methods including potential efficacy under in-use conditions, recommendations for use and labelling in the whole field of chemical disinfection and antiseptics. Areas of activity include agriculture (but not crop protection chemicals), domestic service, food hygiene and other industrial fields, institutional, medical and veterinary applications. Following a meeting in 1990, the Technical Committee (TC) delegated its work to four Working Groups (WG); a Horizontal Working Group (HWG) and three Working Groups responsible for the Medical (WG1), Veterinary (WG2) and Food Hygiene, Domestic and Institutional (WG3) market sectors. Whilst the three WGs could develop test methods to assess bactericidal and fungicidal product activity, specialist Task Groups of the HWG have been established to provide specific guidance on viruses, spores, surface tests, ring trials and to harmonize methodology. The main objective of TC/216 is to produce test methods in a sequential, three phase mode. In Phase 1, the ability of a product to demonstrate bactericidal, fungicidal or sporicidal activity is tested. Phase 2 tests are divided into two steps. Step 1 tests are suspension tests to determine bactericidal, fungicidal, virucidal or sporicidal activity under laboratory conditions that simulate practical conditions. Step 2 tests are other laboratory tests e.g. handwash, handrub or surface tests that are more representative of in-use conditions. Phase 3 tests hope to give guidance to product users as to how to undertake suitable field trials. To date, eight standards have been produced and another 18 are in the final stages of development. WG1 has been the most prolific with 18 test methods under development whilst WG2 and 3 have six and three tests, respectively. Following production of standard test methodologies, the major issues for CEN/TC 216 are concerned with assessing the performance of the tests in practice, especially their statistical reliability. In addition, standards are being further harmonized and guidelines being developed to help product manufacturers and users select the appropriate tests for appropriate fields of use.     

Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.     

Structure and evolution of a family of genes encoding antiseptic and disinfectant resistance in Staphylococcus aureus.

Resistance to antiseptics and disinfectants in Staphylococcus aureus, encoded by the qacC/qacD gene family, is associated with genetically dissimilar small, nontransmissible (pSK89) and large conjugative (pSK41) plasmids. The qacC and qacD genes were analysed in detail through deletion mapping and nucleotide sequence analysis, and shown to encode the same polypeptide, predicted to be 107 aa in size. Direct repeat elements flank the qacD gene, elements which also flank the qacC gene in truncated forms. These elements contain palA sequences, regions of DNA required for replication of some plasmids in S. aureus. The qacC gene is predicted to have evolved from the qacD gene, and in the process to have become reliant on new promoter sequences for its expression. The entire sequence of the 2.4-kb plasmid pSK89 (which contains qacC) was determined, and is compared with other plasmids from Gram + bacteria.     

Development of reproducible test inocula for disinfectant testing

For the development and approval of disinfectants, laboratory tests which combine repeatability and reproducibility with relevance to practical conditions are required to ensure optimum standards of efficacy under use conditions. Over the years, although an increasingly rigorous approach has been adopted in devising European and US Standard Test Methods for disinfectants, which includes specifying all aspects of test methodology, the precision of these methods remains a matter for concern. Studies of proposed European test methods indicate that, although some of the variability is methodological in origin, or is derived from errors in preparing test solutions and performing the tests, one of the major sources of error is lack of reproducibility in the performance of the test inoculum. Results indicate that day to day variability in biocide sensitivity of sequential subcultures arises not only from variations in the phenotype generated from the laboratory stock culture, but also from lack of standardization of conditions used to harvest and prepare test inocula. Further reductions in reproducibility between test periods both within or between laboratories probably arise from alterations in the genotype of the laboratory stock culture or source culture during storage under conditions of refrigeration, freezing or freeze-drying. Methods for production of reproducible inocula are considered and some studies aimed at development of test inocula with more reproducible biocide resistance are described.     

Comparative evaluation of biofilm disinfectant efficacy tests

Regulatory agencies are receiving registration applications for unprecedented, antibiofilm label claims for disinfectants. Reliable, practical, and relevant laboratory biofilm test methods are required to support such claims. This investigation describes the influence of fluid dynamics on the relevancy of a laboratory test. Several disinfectant formulations were tested using three different biofilm testing systems run side-by-side: the CDC biofilm reactor system that created turbulent flow (Reynolds number between 800 and 1850), the drip flow biofilm reactor system that created slow laminar flow (Reynolds number between 12 and 20), and the static biofilm system that involved no fluid flow. Each comparative experiment also included a dried surface carrier test and a dried biofilm test. All five disinfectant tests used glass coupons and followed the same steps for treatment, neutralization, viable cell counting, and calculating the log reduction (LR). Three different disinfectants, chlorine, a quaternary ammonium compound, and a phenolic, were each applied at two concentrations. Experiments were conducted separately with Pseudomonas aeruginosa and Staphylococcus aureus and every experiment was independently repeated. The results showed that biofilm grown in the CDC reactor produced the smallest LR, the static biofilm produced the largest LR, and biofilm grown in the drip flow reactor produced an intermediate LR. The differences were large enough to be of practical importance. The dried surface test often produced a significantly higher LR than the tests against hydrated or dried biofilm. The dried biofilm test produced LR values similar to those for the corresponding hydrated biofilm test. These results show that the efficacy of a disinfectant must be measured by using a laboratory method where biofilm is grown under fluid flow conditions similar to the environment where the disinfectant will be applied.     

Topology testing of phylogenies using least squares methods

Background  

The least squares (LS) method for constructing confidence sets of trees is closely related to LS tree building methods, in which the goodness of fit of the distances measured on the tree (patristic distances) to the observed distances between taxa is the criterion used for selecting the best topology. The generalized LS (GLS) method for topology testing is often frustrated by the computational difficulties in calculating the covariance matrix and its inverse, which in practice requires approximations. The weighted LS (WLS) allows for a more efficient albeit approximate calculation of the test statistic by ignoring the covariances between the distances.     

几种常用防腐剂对日化产品中腐败微生物抑制效果研究

了解几种常用防腐剂在日化产品基质中的防腐效果。采用最低抑菌浓度方法测试卡松、对羟基苯甲酸甲酯、苯氧乙醇、咪唑烷基脲、苯甲醇对腐败微生物的抑菌活性;采用微生物挑战性实验验证卡松、对羟基苯甲酸甲酯、咪唑烷基脲、苯氧乙醇和苯甲醇的抑菌效果。日化产品中分离的腐败微生物对卡松耐受性接近0.0015%,接近《化妆品卫生规范》2007版的限定,其它4种防腐剂在规定的使用范围内对分离的微生物都具有较好的抑制效果;卡松剂量在0.0015%、对羟基苯甲酸甲酯剂量在0.2%、咪唑烷基脲剂量在0.075%、苯甲醇剂量在0.2%时日化产品能通过微生物挑战。日化企业应根据实际情况筛选、复配防腐剂,建立更加高效、经济的防腐体系。     

三种不同方法检查尿液有形成分结果的比较研究

目的:探讨利用三种不同方法检查尿液有形成分的符合率及其影响因素.方法:采用AX-4280全自动干化学分析仪、IQ200全自动尿液分析仪和人工镜检法联合检测尿液,并对结果进行比较和分析.结果:尿液各种有形成分的检测结果均存在不同程度的假阳性,人工镜检法与AX-4280的符合率为:红细胞71.1%;白细胞76.4%.人工镜检法与IQ200全自动尿液分析仪人工修饰前和人工修饰后结果的符合率分别为:红细胞81.3%、91.5%;白细胞85.9%、93.8%;透明管型81.4%、89.7%,未分类管型52.6%、77.4%:结晶84.1%、92.4%;细菌65.1%、88.2%;精子72.0%、91.5%.以人工镜检为标准,IQ200全自动尿液分析仪人工修饰前和人工修饰后各检测项目的假阳性率分别为:红细胞23.0%、9.3%,白细胞16.4%、6.6%,透明管型22.9%、11.4%,未分类管型90.2%、29.2%.结晶19.004、8.2%,细菌53.7%、11.0%,精子38.9%、9.3%.结论:三种检查方法的联合应用更能提高尿液有形成分检查结果的准确性,为临床诊治提供依据.     

三种检验方法在诊断女性生殖道沙眼衣原体感染的比较研究

目的:比较不同方法对宫颈拭子样本沙眼衣原体的检测效果.方法:采集300例20-50岁无感染症状女性宫颈分泌物,分别采用细胞培养、聚合酶链式反应及胶体金法进行沙眼衣原体检测,培养阳性或两个非细胞培养方法检测为阳性,定为真阳性,称为"扩大金标准".结果:300例受检查者.按"扩大金标准"CT感染者共36例,发病率为12%(36/300),细胞培养、聚合酶链式反应、胶体金法敏感性分别为72.22%、91.67%、52.78%.特异性分别为100%、98.11%、98.86%.阳性预测值分别为100%、86.84%、86.36%.阴性预测值分别为96.35%、98.85%、93.88%.结论:三种检测方法均可用于CT感染的诊断.     

Gene delivery in adherent and suspension cells using the combined physical methods

Cytotechnology - Physical methods are widely utilized to deliver nucleic acids into cells such as electro-transfection or heat shock. An efficient gene electro-transfection requires the best...     

Evaluation of the repeatability and reproducibility of European suspension test methods for antimicrobial activity of disinfectants and antiseptics

A collaborative study to determine the precision of the 1987 Method of test for the antimicrobial activity of disinfectants in food hygiene is described. The repeatability and reproducibility of the test was found to vary according to the nature of the test organism, the type of disinfectant product and the skill of the operator. Results indicate that significant differences in microbicidal effect (ME values) occur within test laboratories between test periods as well as between laboratories, and that much of this variability derives from apparently random variations in the resistance of test strains from day to day and test period to test period. Indications are that although the test is sufficiently reliable to be used as a standard method, adequate test replication must be specified to distinguish borderline pass from borderline fail disinfectant concentrations. The implications of the results in relation to current work on the development of unified European test methods for disinfectants is discussed.     

The development and standardization of testing methods for genetically modified organisms and their derived products

As the worldwide commercialization of genetically modified organisms (GMOs) increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.     

Biotechnology products and European consumers

More than 100 interviews conducted during 1997 with European food manufacturers and retailers, trade associations, government departments, consumer groups, environmental organizations and some individual academic scientists revealed how differences in the perceived attitudes of consumers gave rise to varying approaches by suppliers to the possible introduction of transgenic foods. European consumers generally are not against the pharmaceutical products of biotechnology but are much less willing to accept food and food ingredients, especially when derived from genetically modified plants. Objections are mainly based on fears for the health and safety of the consumer, worries about the possibility of deleterious effects on the environment, and a range of moral and ethical concerns often deriving from a distaste, however expressed, at the concept of interfering with nature. Consumer understanding of the science underlying biotechnology is patchy; in no country does more than a small proportion of the population claim a good grasp. Partly no doubt as a consequence of these attitudes, the introduction of genetically modified foods into Europe has occurred slowly and, during the period of this study, perhaps only in the Netherlands and the UK.