Standardized nonculture techniques recommended for European reference laboratories

Culture-confirmed diagnosis of meningococcal invasive infections is often hindered by early antibiotic treatment. Nonculture molecular standardized methods are now essential tools for the immediate management of meningococcal infections. The European Monitoring Group on Meningococci (EMGM) recommends the following measures. (1) The implementation of standardized protocols of extraction methods for DNA isolation from clinical specimens for PCR-based identification and genogrouping of Neisseria meningitidis. (2) The use of molecular approaches (sequencing of target genes) for the determination of meningococcal susceptibility to antibiotics, such as sequencing of penA and rpoB genes for susceptibility to penicillin G and rifampicin, respectively. (3) The use of nonculture strain characterization by multilocus sequence typing (MLST) and sequence typing of porA and fetA. These approaches can be implemented either by individual reference laboratories or through collaboration and referral between centres.     

Molecular typing of meningococci: recommendations for target choice and nomenclature

The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci-these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32).     

HLA techniques: typing and antibody detection in the laboratory of immunogenetics

The HLA loci are a part of the genetic region known as the major histocompatibility complex (MHC). In the last twenty years there has been an exponential growth in the application of DNA technology to the field of histocompatibility and immunogenetics. Histocompatibility between the patient and donor is a prerequisite for the success of haematopoietic stem cell transplantation. In haematopoietic stem cell transplantation allele-level typing needs to evaluate compatibility for the HLA-A,B,C Class I and DRB1 and DQB1 Class II loci in the average transplant program because it is well established that mismatches at certain HLA loci between donor-recipients are closely linked to the risk of graft versus host disease. Resolution at an antigen level in solid organ transplantation is currently sufficient for HLA-A,B and DR antigens and it could be achieved by serological or molecular biology techniques. In solid organ transplantation the definition of antibodies in the recipient to HLA antigens is more important and it was performed primarily by serological technique and more recently by solid phase immunoassays that are more sensitive and specific.     

Structural and antigenic differences between two types of meningococcal pili

Abstract Strains of meningococci, which were shown to be pilated by electron microscopy, could be divided into two groups on the basis of antigenicity and subunit M _r. Strains from group 1 which reacted with monoclonal antibodies directed against gonococcal pili, had pili with subunit M _r similar to that of gonococci which could be detected by radioimmune precipitation or electroblotting. Strains from group 2 failed to react with the monoclonal antibodies and had pili with lower subunit M _r which could only be detected by radioimmune precipitation using polyclonal antipilus antiserum and not by electroblotting.     

PCR for the detection and typing of campylobacters

The flaA gene of Campylobacter sp. was amplified using PCR. Primers were chosen which amplified 1.3 kb of the flaA gene in Camp. jejuni and Camp. coli. 'Campylobacter upsaliensis' amplimer was approximately 1.7 kb in size and was easily distinguishable. Other species of campylobacter failed to yield amplimer. The amplimer was digested with Alu 1 which demonstrated considerable restriction fragment length polymorphism and should allow the development of a rapid novel typing scheme which does not rely on previous culture of campylobacter strains.     

Comparison of Cervical Cytology Reporting Rates: A Useful Adjunct to External Quality Assurance

Reporting rates for abnormal and inadequate cervical smears have been compared during a 5-year period in nine laboratories and related to targets and achievable ranges recently recommended by the National Health Service Cervical Screening Programme (NHSCSP). There was improved consistency in rates for all grades of abnormality as well as inadequate smears. the average rate for moderate and severe dyskaryosis combined increased to 1.6%, which is the target recommended by the NHSCSP, and the standard deviation fell from 0.52 to 0.27. Although average rates for mild dyskaryosis and borderline nuclear change combined and inadequate smears both remained within the achievable range recommended by the NHSCSP, four laboratories were above the upper limit for each of those categories in the final year. During the 5 years of the study there was a fall in the number of ‘inflammatory’smears coded as negative with a recommendation for early repeat. Only one laboratory still uses that category. the challenge for the future lies in controlling high rates for minor abnormalities and inadequate smears while maintaining acceptable rates for moderate and severe dyskaryosis. Comparison of reporting rates is regarded by the participating laboratories as a useful adjunct to external quality assurance. Les taux des frottis cervico-utérins anormaux et inadéquats ont été comparés sur une période de cinq ans pour neuf laboratoires et évalues par rapport aux objectifs et aux intervalles de valeur récemment publiés dans les recommandations du programme de dépistage cervico-utérin du NHS (NHSCSP). Une amélioration de l'homogenéïté des taux de tous les grades d'anomalies aussi bien que celui des frottis inadéquats a été observée. Les taux moyens de toutes les catégories ont augmentés. Le taux moyen de I'ensemble des dyscaryoses modérées et sévères, a augmenté pour atteindre 1,6%, ce qui est I'objectif recommandé par le NHSCSP, et I'ecart type est passé de 0,52 à 0,27. Bien que les taux moyens de l'ensemble des dyscaryoses légéres et des modifications nucléaires “borderline” (BNC) sgient restés dans l'intervalle recommandé par le NHSCSP, quatre laboratoires étaient audessus de la limite supérieure pour chacune de ces catégories, au cours de la derniére année. Au cours des cinq années de cette étude, il y a eu une chute du nombre des “frottis inflammatoires” codés comme négatifs mais où un contrôle rapproaché avait été recommandé. Un seul laboratoire utilise encore cette catégorie. Pour le futur, le challenge se situe au nivau du contrôle des taux élevés d'anomalies mineures et de frottis inadéquats tout en maintenant des taux acceptables de lésions de dyscaryoses moyennes à sévères. La comparaison des taux des différentes catégories de frottis est considérée comme une aide très utile pour I'assurance de qualité externe pour les différents laboratoires participants. Der Anteil abnormer und nicht verwertbarer Abstriche wurde über einen Zeitraum von fünf Jahren zwischen neun Labors und den Empfehlungen des National, Health Service Cervical Screening Programme (NHSCSP) verglichen. Es ergab sich eine bessere Übereinstimmung für alle Anomalien sowie die Rate nichtauswertbarer Abstriche. Der Anteil mässinger und schwerer Dysplasien zusammengefasst stieg auf 1,6% und entsprach damit der Vorgabe des NHSCSP. Die Standardab-weichung sank von 0,52 auf 0,27. Obgleich auch der Anteil der leichten Dysplasien und borderline-Fälle im Bereich der NHSCSP-Empfehlungen blieben lagen dennoch vier Labors auch im letzten Jahr oberhalb der Grenzwerte. Im Untersuchungszeitraum sank die Zahl der als enzündlich beurteilten und zur Kontrolle empfohlenen Fälle. Nur eines der Labors benützt diese Beurteilung noch. Die Forderung der Zukunft wird die Senkung der nichtauswertbaren Präparate und leichten Dysplasien unter Beibehaltung des Standards für mässige und schwere Dysplasien sein. Der Vergleich der Beurteilungen zwischen Labors wird als nützliche Ergänzung der externen Qualitätssicherung angesehen.     

On the evaluation of edgeless diode detectors for patient-specific QA in high-dose stereotactic radiosurgery

PurposeIn this work, the potential of an innovative “edgeless” silicon diode was evaluated as a response to the still unmet need of a reliable tool for plan dosimetry verification of very high dose, non-coplanar, patient-specific radiosurgery treatments. In order to prove the effectiveness of the proposed technology, we focused on radiosurgical treatments for functional disease like tremor or pain.MethodsThe edgeless diodes response has been validated with respect to clinical practice standard detectors by reproducing the reference dosimetry data adopted for the Treatment Planning System. In order to evaluate the potential for radiosurgery patient-specific treatment plan verification, the anthropomorphic phantom Alderson RANDO has been adopted along with three edgeless sensors, one placed in the centre of the Planning Target Volume, one superiorly and one inferiorly.ResultsThe reference dosimetry data obtained from the edgeless detectors are within 2.6% for output factor, off-axis ratio and well within 2% for tissue phantom ratio when compared to PTW 60,018 diode. The edgeless detectors measure a dose discrepancy of approximately 3.6% from the mean value calculated by the TPS. Larger discrepancies are obtained in very steep gradient dose regions when the sensors are placed outside the PTV.ConclusionsThe angular independent edgeless diode is proposed as an innovative dosimeter for patient quality assurance of brain functional disorders and other radiosurgery treatments. The comparison of the diode measurements with TPS calculations confirms that edgeless diodes are suitable candidates for patient-specific dosimetric verification in very high dose ranges delivered by non-isocentric stereotactic radiosurgery modalities.     

分子分型技术用于流脑疫情的同源性分析

目的 应用多位点序列分型（Multilocus sequence typing,MLST）技术和脉冲场凝胶电泳（pulsed-field gel electrophoresis,PFGE）技术对大连市2014年4月同一工地两起流脑疫情得到的脑膜炎奈瑟氏菌株进行分子分型图谱分析,了解各菌株之间的亲缘关系。方法 对病例的脑脊液标本进行脑膜炎奈瑟菌PCR分群,对另一病例和所有密切接触者分离菌株进行多位点序列分型（MLST）试验和脉冲场凝胶电泳（PFGE）鉴定实验。结果 疑似病例标本PCR结果为脑膜炎奈瑟氏菌A群阳性,另一病例和所有密切接触者分离到的菌株PFGE图谱基本相同,表明来自同一克隆系。多位点序列分析结果为ST7。结论 两起疫情分离到的菌株型别之间具有高度的同源性,该病原菌类型为ST7的A群脑膜炎奈瑟氏菌。     

Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates

In this study, we compared the potential of amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of tandem repeats (VNTR) of the P97 encoding gene, as possible methods for typing an international collection of Mycoplasma hyopneumoniae isolates. All techniques showed a typeability of 100% and high intraspecific diversity. However, the discriminatory power of the different techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene (>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97 (<0.92). Other, preferentially well spread, tandem repeat regions should be included in order for this latter technique to become valuable for typing purposes. RAPD was also found to be a less interesting typing technique because of its low reproducibility between different runs. Nevertheless, all molecular techniques showed overall more resemblance between strains isolated from different pigs from the same herd. On the other hand, none of the techniques was able to show a clear relationship between the country of origin and the fingerprints obtained. We conclude that AFLP and an earlier described PFGE technique are highly reliable and discriminatory typing techniques for outlining the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an equally useful alternative for demonstrating intraspecific variability, although the generation of sequence variability of the gene remains unclear and must be further examined.     

Qualitative PCR-ELISA protocol for the detection and typing of viral genomes

PCR is an established technique providing rapid and highly productive amplification of specific DNA sequences. The demand for equally rapid, sensitive and objective methods to achieve detection of PCR products has led to the coupling of PCR with ELISA. PCR-ELISA involves direct incorporation of labeled nucleotides in amplicons during PCR-amplification, their hybridization to specific probes and hybrid capture-immunoassay in microtiter wells. PCR-ELISA is performed in 1 d and is very flexible, with the ability to process simultaneously up to 96 or 384 samples. This technique is potentially automatable and does not require expensive equipment, and thus can be fundamental in laboratories without access to a real-time PCR thermocycler. PCR-ELISA has mainly been used to detect infectious agents, including viruses, bacteria, protozoa and fungi. A PCR-ELISA protocol for the qualitative detection of papillomavirus genomes and simultaneous typing of different genotypes are detailed here as an example of the technique.     

基于DRGs管理工具的医疗质量持续改进

近年来医疗技术水平迅速发展,医疗技术管理的水平也随之快速发展。愈来愈多的国内外先进的医学管理理念不断运用到实践,其中在国外目前广泛运用的DRGs诊断相关分组系统近几年在国内也不断推广试点。我院作为北京首批试点医院,经过近5年的试点,在医疗质量、效率、安全等各方面都有明显的改善,DRGs是一种行之有效的促进医疗管理持续改进的管理工具。     

国外诊断相关组付费制度与医疗服务质量关系研究述评

通过理论分析和文献综述,介绍此付费制度与医疗服务质量关系的研究现状,探讨了一些国家通过引入医疗机构提供能力、医疗服务质量评价标准及全面质量管理观念等措施,对制度进行改良的实践效果,对我国医疗付费制度的改革和优化具有重要的启示意义。     

Application of PCR, rep-PCR and RAPD techniques for typing ofLactococcus lactis strains

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.     

PCR-测序法对啮齿类动物汉坦病毒的检测和基因分型

目的探讨PCR-测序法在啮齿类动物汉坦病毒临床检测中的应用价值。方法以Genbank 7种血清亚型24株汉坦病毒代表性毒株为基础,从病毒基因S片段序列设计引物,采用邻位相连法进行系统进化分析,以该方法对浙江省近年从野生啮齿类动物中临床分离的汉坦病毒毒株进行分型鉴定。结果所建系统发育树将所分析的毒株分为5个区域,引起HFRS的四种血清型具有较稳定的拓扑结构,且能与引起HPS的血清型进行区分。11株实验毒株进行PCR扩增和测序,结果表明该引物具有高度的敏感性和特异性,其中9株浙江省分离的血清型未知毒株的系统发育分析发现其包含引起HFRS的3株HTN和1株SEO血清型,其他5株属两种未知血清型。讨论所建立的PCR-测序法具有用于临床检测汉坦病毒的价值。     

Comparison of pre-treatment and in-vivo dosimetry for advanced radiotherapy of prostate cancer

BackgroundThe usage of advanced radiotherapy techniques requires validation of a previously calculated dose with the precise delivery with a linear accelerator. This study aimed to review and evaluate new verification methods of dose distribution. Moreover, our purpose was to define an internal protocol of acceptance for in-vivo measurements of dose distribution.Materials and methodsThis study included 43 treatment plans of prostate cancer calculated using the Monte Carlo algorithm. All plans were delivered using the Volumetric Modulated Arc Therapy (VMAT) technique of advanced radiotherapy by the linear accelerator Elekta VersaHD. The dose distribution was verified using: MatriXX, iViewDose, and in-vivo measurements. The verification also included recalculation of fluence maps of quality assurance plans in another independent algorithm.ResultsThe acceptance criterion of 95% points of dose in agreement was found for pre-treatment verification using MatriXX; the average γ value was 99.09 ± 0.93 (SD) and 99.64 ± 0.35 (SD) for recalculation in the Collapse Cone algorithm. Moreover, using the second algorithm in the verification process showed a positive correlation ρ = 0.58, p < 0.001. However, verification using iViewDose in a phantom and in-vivo did not meet this γ-pass rate.ConclusionsEvaluation of gamma values for in-vivo measurements utilizing iViewDose software was helpful to establish an internal dosimetry protocol for prostate cancer treatments. We assumed value at a minimum of 50% points of the dose in agreement with the 3%/3 mm criterion as an acceptable compliance level. The recalculated dose distribution of QA plans in regard to the Collapse Cone algorithm in the other treatment planning system can be used as a pre-treatment verification method used by a medical physicist in their daily work. The effectiveness of use in iViewDose software, as a pre-treatment tool, is still debatable, unlike the MatriXX device.     

TomoEQA: Dose verification for patient-specific quality assurance in helical tomotherapy using an exit detector

PurposeExisting phantom-less quality assurance (QA) platforms does not provide patient-specific QA for helical tomotherapy (HT). A new system, called TomoEQA, is presented to facilitate this using the leaf open time (LOT) of a binary multi-leaf collimator, as measured by an exit detector.MethodsTomoEQA was designed to provide measurement-based LOTs based on detector data and to generate a new digital imaging and communication in medicine (DICOM) dataset that includes the measured LOTs for use by secondary check platforms. To evaluate the system, 20 patient-specific QAs were performed using the program in Mobius3D software, and the results were compared to conventional phantom-based QA results.ResultsFrom our assessment, most of the differences between the planned and measured (or calculated) data, excluding one case, were within the acceptance criteria comparing with those of conventional QA. Regarding the gamma analysis, all results considered in this study were within the acceptance criteria. In addition, the developed system was performed for a failed case and showed approximately the same trends as the conventional approach.ConclusionsTomoEQA could perform patient-specific QAs of HT using Mobius3D and provide reliable patient-specific QAs results by evaluating point dose errors and 3D gamma passing rates. TomoEQA could also distinguish whether an intensity-modulated radiation therapy plan failed or not.