A rapid PFGE protocol for typing Legionella isolates from fresh or frozen samples

Within the realm of studies on nosocomial infections and epidemiology, pulsed field gel electrophoresis (PFGE) is often considered as the “gold standard” for typing of Legionella or other bacteria. Performing this protocol usually requires 2–5 days; this excessive time requirement, lack of a standardized procedure, and high cost have limited its use. However, recently the typing of Legionella with PFGE has been reduced to about 2 days, and we further shortened the procedure by reducing the time for the plug preparation and electrophoresis steps. To shorten plug preparation, we used a strong thermal shock and high-temperature washes to reduce cell lysis and DNA isolation time, and we stressed the electrophoresis to obtain comparable macrorestriction patterns among strains in 16 h. The DNA digestion phase was not altered. We also applied this protocol directly to frozen bacteria from strain collections with the aim of shortening the entire procedure. We developed a protocol that can be completed in 24 h, or less if necessary, while avoiding some typical artifacts of traditional procedures. This new protocol also provides good results when directly applied to frozen material from strain collections, thus saving bacteria growth time. Our observations indicate that PFGE tolerates a wide range of adjustments, allowing its application to fresh or frozen samples in a short amount of time.     

Reduced mortality after the implementation of a protocol for the early detection of severe sepsis

Objective

We evaluate the impact that implementing an in-hospital protocol for the early detection of sepsis risk has on mortality from severe sepsis/septic shock.

Methods

This was a prospective cohort study conducted in 2 phases at 2 general hospitals in Brazil. In phase I, patients with severe sepsis/septic shock were identified and treated in accordance with the Surviving Sepsis Campaign guidelines. Over the subsequent 12 months (phase II), patients with severe sepsis/septic shock were identified by means of active surveillance for signs of sepsis risk (SSR). We compared the 2 cohorts in terms of demographic variables, the time required for the identification of at least 2 SSRs, compliance with sepsis bundles (6- and 24-hour), and mortality rates.

Results

We identified 217 patients with severe sepsis/septic shock (102 during phase I and 115 during phase II). There were significant differences between phases I and II in terms of the time required for the identification of at least 2 SSRs (34 ± 48 vs 11 ± 17 hours; P < .001) and in terms of in-hospital mortality (61.7% vs 38.2%; P < .001).

Conclusion

The early detection of sepsis promoted early treatment, reducing in-hospital mortality from severe sepsis/septic shock.     

Heat-shock treatment in a rapid assay for cytomegalovirus detection in clinical samples

A mild heat-shock treatment of cells enhances cytomegalovirus (CMV) infection and shortens its eclipse period. This provided the basis for the development of a rapid assay to detect CMV in clinical samples.Urine specimens were inoculated in heat-shocked cells and then CMV-infected cells were visualized at various hours after infection with a monoclonal antibody directed against a CMV-induced immediate-early protein, using an immunoalkaline phosphatase staining. Out of 104 urine specimens examined, 13 proved positive and the assay we describe was able to yield positive results in a single day.     

A fluorescence polarization immunoassay for detection of thiacloprid in environmental and agricultural samples

As a widely used neonicotinoid insecticide, thiacloprid has been observed to pose a risk to honeybees and the endocrine system of mammals. So a detection method with high sensitivity, simple operation and high throughput is required. Based on this consideration, we prepared an anti-thiacloprid monoclonal antibody (mAb, C9) and developed a fluorescence polarization immunoassay (FPIA) for the detection of thiacloprid. After optimizing the length of spacer and reaction conditions, the 50% inhibition concentration (IC₅₀), limit of detection (LOD) and linear range (IC₂₀ ∼ IC₈₀) of the FPIA are 15.34 ng mL⁻¹, 2.43 ng mL⁻¹ and 3.10–65.7 ng mL⁻¹, respectively. Meanwhile, FPIA just requires 12 min to detect the pesticide with simple operation. Then the FPIA was used to detect the thiacloprid in spiked rice, soil, cucumber and tomato samples, and recoveries were in the range of 79.1%–105.3% with 3.7%–12.3% standard deviation. The FPIA also shows good correlation with high-performance liquid chromatography for the detection of thiacloprid in tomato samples.

An anti-thiacloprid monoclonal antibody with high sensitivity was prepared and used to develop a fluorescence polarized immunoassay.     

An rpoD-based PCR procedure for the identification of Pseudomonas species and for their detection in environmental samples

A polymerase chain reaction-based approach was developed for species identification of Pseudomonas strains and for the direct detection of Pseudomonas populations in their natural environment. A highly selective set of primers (PsEG30F and PsEG790R), giving an amplicon of 760 nucleotides in length, was designed based on the internal conserved sequences of 33 selected rpoD gene sequences (the sigma 70 factor subunit of the DNA polymerase) of Pseudomonas type strains, representing the entire intrageneric phylogenetic clusters described in the genus. The utility of the primer set was verified on 96 Pseudomonas type strains and on another 112 recognised Pseudomonas strains. The specificity of the primer set was also tested against strains from species not belonging to the genus Pseudomonas. These primers were also shown to be useful for the direct detection of Pseudomonas species in environmental DNA after a cloning procedure. These results were compared in parallel with other cloning procedures described previously, based on the analysis of other genes (16S rDNA and ITS1) and also by using primers designed for rpoD on sequences from gamma-proteobacteria. All of the cultured Pseudomonas strains tested could be amplified with these novel primers, indicating that this method is also a useful tool for the specific analysis of Pseudomonas populations from environmental samples without the need for cultivation.     

Recommendations for detection and management of unsuitable samples in clinical laboratories.

A large body of evidence attests that quality programs developed around the analytical phase of the total testing process would only produce limited improvements, since the large majority of errors encountered in clinical laboratories still prevails within extra-analytical areas of testing, especially in manually intensive preanalytical processes. Most preanalytical errors result from system flaws and insufficient audit of the operators involved in specimen collection and handling responsibilities, leading to an unacceptable number of unsuitable specimens due to misidentification, in vitro hemolysis, clotting, inappropriate volume, wrong container or contamination from infusive routes. Detection and management of unsuitable samples are necessary to overcome this variability. The present document, issued by the Italian Inter-society SIBioC-SIMeL-CISMEL (Society of Clinical Biochemistry and Clinical Molecular Biology-Italian Society of Laboratory Medicine-Italian Committee for Standardization of Hematological and Laboratory Methods) Study Group on Extra-analytical Variability, reviews the major causes of unsuitable specimens in clinical laboratories, providing consensus recommendations for detection and management.     

Evaluation of Duopath Legionella kit for the rapid identification of Legionella strains isolated from water samples

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a rapid and simple immunochromatographic assay kit for the identification of Legionella species. We evaluated the precision of the kit in identifying 100 strains of Legionella and 35 strains of non-Legionella bacteria isolated from cooling tower and bath water samples. Consequently, of all the Legionella strains tested, 99 strains were judged to be Legionella, and only one strain (Legionella busanensis) was judged to be non-Legionella. All of the 35 non-Legionella strains were judged to be non-Legionella. We therefore conclude that Duopath Legionella is a useful method for the rapid identification of Legionella.     

噬菌体生物扩增法检测临床标本中结核分枝杆菌的系统评价

目的采用Meta分析的方法评价噬菌体生物扩增法检测临床标本中结核分枝杆菌的准确性。方法通过检索PubMed、EMbase、web of science、Cochrane等数据库和其他方式广泛收集文献。根据QUADAS质量评价标准评价纳入文献的质量,应用Meta-Disc软件对噬菌体生物扩增法检测临床标本中结核分枝杆菌的敏感性、特异性、诊断比数比进行合并分析,并进行异质性检验,对纳入研究绘制SROC曲线。结果最终纳入19篇相关文献,共计20个研究。综合分析,合并特异性为96%（96-97%）;合并敏感度为69%（67-72%）;合并诊断比数比为52.30（25.18-108.65）;SROC曲线下面积为0.93。结论对纳入的研究分析证实,噬菌体检测法检测临床标本中的结核分枝杆菌的敏感性偏低,但具有高度特异性,表明该方法在快速诊断结核病的准确性方面具有优势。     

Conscious sedation of pediatric oncology patients for painful procedures: Development and implementation of a clinical practice protocol

Pain during treatment procedures is often identified as the most distressing aspect of the entire cancer experience for a child with cancer. The use of conscious sedation may reduce this procedure-related pain and distress. A clinical practice protocol is necessary to maximize the efficacy and safety of conscious sedation use. In this article, the processes of developing and implementing a conscious sedation protocol for pediatric oncology patients undergoing painful procedures are presented. The protocol itself is included. A review of the literature on conscious sedation is presented, and implications for pediatric oncology nursing practice are discussed.     

Utilization of a plasticized PVC optical sensor for the selective and efficient detection of cobalt(ii) in environmental samples

A novel sensitive, selective, and reversible cobalt(ii) ion optical sensor was prepared by the incorporation of 5-[o-carboxyphenylazo]2,4-dihydroxybenzoic acid [CPDB] and sodium tetraphenylborate (NaTPB) in a plasticized polyvinyl chloride (PVC) membrane containing dioctyl adipate (DOA) as a plasticizer. The influence of several parameters such as pH, base matrix, solvent mediator and reagent concentration was optimized. A comparison of the obtained results with those of previously reported sensors revealed that the proposed method, in addition to being fast and simple, provided a good linear range (0.05–45.20 μM) and low detection limit (0.015 μM). Low detection and quantification limits and excellent selectivity in the presence of interfering ions such as Fe³⁺, Cu²⁺, Ni²⁺, Ag⁺, Au³⁺, Cr³⁺, Cd²⁺, Zn²⁺, Hg²⁺, and SO₄²⁻ make it feasible to monitor Co²⁺ ion content accurately and repeatedly in environmental samples with complicated matrices. The optode was regenerated successfully using 0.3 M nitric acid (HNO₃) solution while its response was reversible with a relative standard deviation (RSD) lower than 1.9% for seven replicate determinations of 20 μM Co²⁺ in various membranes. The optode was stable and was stored for at least 15 days without observing any change in its sensitivity.

A novel sensitive, selective, and reversible cobalt(ii) ion optical sensor was prepared by the incorporation of [CPDB] and (NaTPB) in a (PVC) membrane containing (DOA) as a plasticizer.     

乙型肝炎病毒表面抗原弱反应性标本的检测及临床意义

目的探讨乙型肝炎病毒表面抗原(HBsAg)弱反应性标本的临床检测对策及其临床意义。方法采用酶联免疫吸附试验(ELISA)试剂Ⅰ、Ⅱ和化学发光法分别检测155例HBsAg初检为弱反应性的标本。结果试剂Ⅰ与化学发光法的阳性符合率为47.1%,阴性符合率为5.2%;试剂Ⅱ与化学发光法的阳性符合率为13.5%,阴性符合率为39.3%;试剂Ⅰ与试剂Ⅱ的阳性符合率为25.2%,阴性符合率为7.1%。结论对于ELISA法检测HBsAg为弱反应性的标本,应分析原因并采用多家试剂及更灵敏的方法进一步复查以确认,最好是进行确认实验。     

Clinical consequences of the implementation of a weaning protocol

Objective To analyze the clinical and economic consequences of the implementation of a weaning protocol in patients mechanically ventilated (MV) for more than 48 h.Design Comparative studySetting General intensive care unit (ICU) in a county hospital covering 360 000 inhabitants.Patients 51 patients weaned by a fixed protocol were studied prospectively and compared with 50 retrospective controls.Measurements The following variables were assessed: Acute Physiology and Chronic Health Evaluation (APACHE) II score, age, cause of respiratory failure, type of extubation (direct extubation or extubation using a weaning technique), number of days on MV before the weaning trial, weaning time, total duration of MV, complications (reintubations and tracheostomies), length of ICU stay, and mortality.Results The groups were comparble in terms of age, APACHE II score, and main cause of acute respiratory failure. Number of days on MV up to the weaning trial were similar in the two groups (8.4±7.7 in the protocol group vs 7.5±5.5 in the control group, NS). Most of the patients (80%) in the protocol group were directly extubated without a weaning technique, unlike the control group (10%) (p<0.01). When a weaning technique was used, the weaning time was similar in both groups (3.5±3.9 days vs 3.6±2.2 days in the control group). Duration of MV was shorter in the protocol group (10.4±11.6 days) than in the control group (14.4±10.3 days) (p<0.05). As a result, the ICU stay was reduced by using the weaning protocol (16.7±16.5 days vs 20.3±13.2 days in the control group,p<0.05). We found no differences in reintubation rate (17 vs 14% in the control group) and need for tracheostomies (2 vs 8% in the control group).Conclusion The implementation of a weaning protocol decreased the duration of MV and ICU stay by increasing the number of safe, direct extubations.Supported in part by grant FIS 93/0590     

Recovery of Legionella species from water samples using an internal method based on ISO 11731: suggestions for revision and implementation

The study aim was to determine retrospectively whether the parallel use of 2 media [buffered charcoal yeast extract (BCYE) and medium of Wadowsky and Yee (MWY)] to isolate Legionella spp. from water samples taken from hospital water supply systems increased the sensitivity of the culture method as compared with methods/protocols in which only seeding on a selective medium is used. We analyzed the results obtained from 931 positive water samples. In 484 of the 931 positive water samples, Legionella spp. was isolated in the presence of other microorganisms; in 83% (400/484), we used MWY to count suspected colonies, which gave a lower number of unreadable plates. In the 447 samples containing only Legionella spp., the highest frequency of positive samples (93%, 418/447) was obtained with BCYE, whereas seeding on MWY yielded 78% (348/447) (P < 0.001). Evaluation of the influence of the media on the Legionella spp. counts obtained by the 2 media showed that BCYE agar produced significantly higher counts than MWY (P < 0.001). The major conclusions that may be drawn from our data are as follows: 1) BCYE gives a high recovery rate of positive samples (93%) and a much greater yield of Legionella spp. than MWY; 2) BCYE was necessary for the detection of non-L. pneumophila spp. which grew poorly on selective media; 3) selective media [MWY or GVPC (glycine, vancomycin, polymyxin B, and cycloheximide)] were necessary for the recovery of Legionella spp. when the non-selective medium (BCYE) was difficult to interpret because of contaminating background flora. The use of different media is recommended for routine water tests in hospitals.     

Evaluation of a novel Chlamydia trachomatis microsphere suspension assay for detection and genotyping of the different serovars in clinical samples

A novel Chlamydia trachomatis (Ct) microsphere suspension (MS) assay was evaluated for identification of the different serovars, using the same PCR primer set established for the Ct Detection and genoTyping assay. Both assays can detect and identify all 14 major serovars (A, B/Ba, C, D/Da, E, F, G/Ga, H, I/Ia, J, K, L1, L2/L2a, and L3) and one genovariant of serovar J. The probe specificity for the Ct-MS assay was determined using 14 Ct reference strains and 1 clinical isolate from a genovariant of serovar J. Also, the Ct-MS assay and the Ct detection and genoTyping assay were compared in 712 Ct-positive clinical samples. The Ct-MS assay showed a highly specific reaction for all probes with the amplicons of the reference strains, giving a very low background median fluorescence intensity signal (median fluorescence intensity ≤ 10). An excellent overall agreement in the Ct detection (kappa = 0.947, 95% confidence interval, 0.89 to 0.999; McNemar's test, P = 1.000) and the Ct genotyping (kappa = 0.993, 95% confidence interval, 0.977 to 1.000; McNemar's test, P = 0.053) was observed between the Ct detection and genoTyping (DT) assay and the Ct-MS assay. In conclusion, the novel Ct-MS assay permits simultaneous detection and genotyping of Ct serovars, making the Ct-MS assay an excellent high throughput method.     

Development of a gold-nanorod-based lateral flow immunoassay for a fast and dual-modal detection of C-reactive protein in clinical plasma samples

Fast and simple detection of C-reactive protein (CRP) is highly significant for the diagnosis and prognosis of inflammatory or infectious diseases. Lateral flow immunoassay has the advantages of rapid detection, simple operation and low cost, but it is usually limited by the quantitative ability and speed of data extraction. Herein, a gold-nanorod-based lateral flow immunoassay was developed to rapidly detect CRP by simultaneously monitoring the colorimetric and temperature signals. In this method, anti-CRP antibody-modified gold nanorods (GNRs) were designed as colorimetric and photothermal conversion probes. A mouse anti-CRP monoclonal antibody and goat anti-mouse IgG were used as test and control lines, respectively. Then, a lateral flow immunochromatographic strip was constructed by a sandwich-type method for detecting CRP by introducing antibody-modified GNRs, and this procedure needed less than 15 min. Finally, the detection signals can be directly observed by eyes and directly read using a thermal imager. The as-synthesized GNR showed high photothermal conversion efficiency (η = 39%) and strong localized surface plasmon resonance (LSPR) absorption. For CRP detection, the proposed immunochromatographic strip exhibited good specificity, high sensitivity, good linearity within the range of 50–10 000 ng mL⁻¹ and a low limit of detection (LOD, 1.3 ng mL⁻¹). This method was successfully applied for CRP detection in clinical plasma samples, and it correlated very well with the diagnostic kit of immunoturbidimetry (r = 0.96). The results indicated that the developed GNR-based immunochromatographic strip has immense potential for use as a rapid and cost-effective in vitro diagnostic kit.

A gold-nanorod-based lateral flow immunoassay for rapid and quantitative detection of CRP by simultaneously monitoring the colorimetric and temperature signals.     

HBsAg阴性及弱反应性标本的检测及临床意义

目的探讨ELISA两步法、化学发光法、荧光定量PCR定量技术对HBsAg阴性及弱反应性标本的检测及其临床意义,为提高临床检测准确率提供借鉴。方法收集医院门诊和住院受检者经ELISA一步法检测的乙肝两对半4种模式标本(模式1:仅HBeAb阳性;模式2:仅HBcAb阳性;模式3:HBcAb阳性和HBeAb阳性;模式4:HBsAg均为弱反应性的标本),用ELISA两步法及化学发光法检测,再对经化学发光法检测HBsAg结果为阳性的标本用FQ-PCR检测乙肝病毒DNA载量。结果仅HBeAb阳性的标本,共2例,ELISA两步法结果显示HBsAg均为弱反应性,化学发光法结果为阴性;仅HBcAb阳性的标本121例,ELISA两步法结果示HBsAg呈弱反应性者为26例(占21.5%),化学发光法结果阳性8例(占16.7%);HBcAb阳性和HBeAb阳性的标本55例,ELISA两步法HBsAg结果呈弱反应性的为25例(占45.4%),化学发光法结果阳性14例(占25.5%),其中7例用常规PCR方法检测结果拷贝数均小于5.0*102;HBsAg为弱反应性的标本59例,ELISA两步法弱反应性的结果均在临界值附近,化学发光法结果均为阳性。结论对于HBsAg阴性及弱反应性的临床标本,最好采用化学发光法检测或者进行确证试验。