Testing of Diagnostic Methods for Detection of Influenza Virusfor Optimal Performance in the Context of an Influenza Surveillance Network

Influenza surveillance networks must detect early the viruses that will cause the forthcoming annual epidemics and isolate the strains for further characterization. We obtained the highest sensitivity (95.4%) with a diagnostic tool that combined a shell-vial assay and reverse transcription-PCR on cell culture supernatants at 48 h, and indeed, recovered the strain

Why Public Health Agencies Cannot Depend on Good Laboratory Practices as a Criterion for Selecting Data: The Case of Bisphenol A

In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES: We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION: Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., "good science"). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS: Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Impact of laboratory cross-contamination on molecular epidemiology studies of tuberculosis.

Laboratory cross-contamination by Mycobacterium tuberculosis is known to be responsible for the misdiagnosis of tuberculosis, but its impact on other contexts has not been analyzed. We present the findings of a molecular epidemiology analysis in which the recent transmission events identified by a genotyping reference center were overestimated as a result of unnoticed laboratory cross-contamination in the original diagnostic laboratories.

The combination of three faecal parasitological methods to improve the diagnosis of schistosomiasis mansoni in a low endemic setting in the state of Ceará, Brazil

Laboratory diagnosis of intestinal schistosomiasis mansoni can be accomplished through various methods of stool examination to detect parasites, ranging from the most classic tests (Kato-Katz) to several methods that are still undergoing validation. This study was conducted to assess two new parasite identification methods for diagnosing schistosomiasis mansoni in residents of a low endemic area in the municipality of Maranguape, in the state of Ceará, Brazil using the Kato-Katz method as a reference and serology (enzyme-linked immunosorbent assay) for the screening of patients. The Kato-Katz, the saline gradient method and the Helmintex® method parasite identification methods were employed only in subjects who exhibited positive serologic tests. The test results were then analysed and treatment of positive individuals was subsequently performed. After comparing the test results, we observed that the saline gradient method and the Helmintex® method were more effective in diagnosing schistosomiasis mansoni in the study area compared with the Kato-Katz method.

The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

In vitro methods for diagnosing nonimmediate hypersensitivity reactions to drugs.

Nonimmediate drug hypersensitivity reactions (DHRs) are difficult to manage in daily clinical practice, mainly owing to their heterogeneous clinical manifestations and the lack of selective biological markers. In vitro methods are necessaryto establish a diagnosis, especially given the low sensitivity of skin tests and the inherent risks of drug provocation testing. In vitro evaluation of nonimmediate DHRs must include approaches that can be applied during the different phases of the reaction. During the acute phase, monitoring markers in both skin and peripheral blood helps to discriminate between immediate and nonimmediate DHRs with cutaneous responses and to distinguish between reactions that, although they present similar clinical symptoms, are produced by different immunological mechanisms and therefore have a different treatment and prognosis. During the resolution phase, in vitro testing is used to detect the response of T cells to drug stimulation; however, this approach has certain limitations, such as the lack of validated studies assessing sensitivity. Moreover, in vitro tests indicate an immune response that is not always related to a DHR. In this review, members of the Immunology and Drug Allergy Committee of the Spanish Society of Allergy and Clinical Immunology (SEAIC) provide an overview of the most widely used in vitro tests for evaluating nonimmediate DHRs.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Study of common database feeding with results coming from different analytical methods in the framework of illicit drugs chemical profiling

Analytical results harmonisation is investigated in this study to provide an alternative to the restrictive approach of analytical methods harmonisation which is recommended nowadays for making possible the exchange of information and then for supporting the fight against illicit drugs trafficking. Indeed, the main goal of this study is to demonstrate that a common database can be fed by a range of different analytical methods, whatever the differences in levels of analytical parameters between these latter ones. For this purpose, a methodology making possible the estimation and even the optimisation of results similarity coming from different analytical methods was then developed. In particular, the possibility to introduce chemical profiles obtained with Fast GC-FID in a GC-MS database is studied in this paper. By the use of the methodology, the similarity of results coming from different analytical methods can be objectively assessed and the utility in practice of database sharing by these methods can be evaluated, depending on profiling purposes (evidential vs. operational perspective tool). This methodology can be regarded as a relevant approach for database feeding by different analytical methods and puts in doubt the necessity to analyse all illicit drugs seizures in one single laboratory or to implement analytical methods harmonisation in each participating laboratory.

The hematology laboratory in blood doping (bd): 2014 update on the athlete biological passport (APB)

Introduction: Blood doping (BD) is the use of Erythropoietic Stimulating Agents (ESAs) and/or transfusion to increase aerobic performance in athletes. Direct toxicologic techniques are insufficient to unmask sophisticated doping protocols. The Hematological module of the ABP (World Anti-Doping Agency), associates decision support technology and expert assessment to indirectly detect BD hematological effects. Methods: The ABP module is based on blood parameters, under strict pre-analytical and analytical rules for collection, storage and transport at 2-12°C, internal and external QC. Accuracy, reproducibility and interlaboratory harmonization fulfill forensic standard. Blood samples are collected in competition and out-ofcompetition. Primary parameters for longitudinal monitoring are: - hemoglobin (HGB); - reticulocyte percentage (RET); - OFF score, indicator of suppressed erythropoiesis, calculated as [HGB(g/L) * 60-√RET%]. Statistical calculation predicts individual expected limits by probabilistic inference. Secondary parameters are RBC, HCT, MCHC-MCH-MCV-RDW-IFR. ABP profiles flagged as atypical are review by experts in hematology, pharmacology, sports medicine or physiology, and classified as: - normal - suspect (to target) - likely due to BD - likely due to pathology. Results: Thousands of athletes worldwide are currently monitored. Since 2010, at least 35 athletes have been sanctioned and others are prosecuted on the sole basis of abnormal ABP, with a 240% increase of positivity to direct tests for ESA, thanks to improved targeting of suspicious athletes (WADA data). Specific doping scenarios have been identified by the Experts (Table and Figure). Figure. Typical HGB and RET profiles in two highly suspicious athletes. A. Sample 2: simultaneous increases in HGB and RET (likely ESA stimulation) in a male. B. Samples 3, 6 and 7: "OFF" picture, with high HGB and low RET in a female. Sample 10: normal HGB and increased RET (ESA or blood withdrawal). Conclusions: ABP is a powerful tool for indirect doping detection, based on the recognition of specific, unphysiological changes triggered by blood doping. The effect of factors of heterogeneity, such as sex and altitude, must also be considered. Schumacher YO, et al. Drug Test Anal 2012, 4:846-853. Sottas PE, et al. Clin Chem 2011, 57:969-976.

Laboratory-based versus non-laboratory-based CVD risk analysis.

Commentaire de: Gaziano TA, Young CR, Fitzmaurice G, Atwood S, Gaziano JM. Laboratory-based versus non-laboratory-based method for assessment of cardiovascular disease risk: the NHANES I Follow-up Study cohort. Lancet. 2008;371(9616):923-31. PMID: 18342687

Laboratory Study of Structural Behavior of Alternative Dowel Bars, April 2006

Load transfer across transverse joints has always been a factor contributing to the useful life of concrete pavements. For many years, round steel dowels have been the conventional load transfer mechanism. Many problems have been associated with the round steel dowels. The most detrimental effect of the steel dowel is corrosion. Repeated loading over time also damages joints. When a dowel is repeatedly loaded over a long period of time, the high bearing stresses found at the top and bottom edge of a bar erode the surrounding concrete. This oblonging creates multiple problems in the joint. Over the past decade, Iowa State University has performed extensive research on new dowel shapes and materials to mitigate the effects of oblonging and corrosion. This report evaluates the bearing stress performance of six different dowel bar types subjected to two different shear load laboratory test methods. The first load test is the AASHTO T253 method. The second procedure is an experimental cantilevered dowel test. The major objective was to investigate and improve the current AASHTO T253 test method for determining the modulus of dowel support, k0. The modified AASHTO test procedure was examined alongside an experimental cantilever dowel test. The modified AASHTO specimens were also subjected to a small-scale fatigue test in order to simulate long-term dowel behavior with respect to concrete joint damage. Loss on ignition tests were also performed on the GFRP dowel specimens to determine the resin content percentage. The study concluded that all of the tested dowel bar shapes and materials were adequate with respect to performance under shear loading. The modified AASHTO method yielded more desirable results than the ones obtained from the cantilever test. The investigators determined that the experimental cantilever test was not a satisfactory test method to replace or verify the AASHTO T253 method.

Developing a Simple and Rapid Test for Monitoring the Heat Evolution of Concrete Mixtures for Both Laboratory and Field Applications, January 2006

Various test methods exist for measuring heat of cement hydration; however, most current methods require expensive equipment, complex testing procedures, and/or extensive time, thus not being suitable for field application. The objectives of this research are to identify, develop, and evaluate a standard test procedure for characterization and quality control of pavement concrete mixtures using a calorimetry technique. This research project has three phases. Phase I was designed to identify the user needs, including performance requirements and precision and bias limits, and to synthesize existing test methods for monitoring the heat of hydration, including device types, configurations, test procedures, measurements, advantages, disadvantages, applications, and accuracy. Phase II was designed to conduct experimental work to evaluate the calorimetry equipment recommended from the Phase I study and to develop a standard test procedure for using the equipment and interpreting the test results. Phase II also includes the development of models and computer programs for prediction of concrete pavement performance based on the characteristics of heat evolution curves. Phase III was designed to study for further development of a much simpler, inexpensive calorimeter for field concrete. In this report, the results from the Phase I study are presented, the plan for the Phase II study is described, and the recommendations for Phase III study are outlined. Phase I has been completed through three major activities: (1) collecting input and advice from the members of the project Technical Working Group (TWG), (2) conducting a literature survey, and (3) performing trials at the CP Tech Center’s research lab. The research results indicate that in addition to predicting maturity/strength, concrete heat evolution test results can also be used for (1) forecasting concrete setting time, (2) specifying curing period, (3) estimating risk of thermal cracking, (4) assessing pavement sawing/finishing time, (5) characterizing cement features, (6) identifying incompatibility of cementitious materials, (7) verifying concrete mix proportions, and (8) selecting materials and/or mix designs for given environmental conditions. Besides concrete materials and mix proportions, the configuration of the calorimeter device, sample size, mixing procedure, and testing environment (temperature) also have significant influences on features of concrete heat evolution process. The research team has found that although various calorimeter tests have been conducted for assorted purposes and the potential uses of calorimeter tests are clear, there is no consensus on how to utilize the heat evolution curves to characterize concrete materials and how to effectively relate the characteristics of heat evolution curves to concrete pavement performance. The goal of the Phase II study is to close these gaps.

The Vaccine Formulation Laboratory: a platform for access to adjuvants.

Adjuvants are increasingly used by the vaccine research and development community, particularly for their ability to enhance immune responses and for their dose-sparing properties. However, they are not readily available to the majority of public sector vaccine research groups, and even those with access to suitable adjuvants may still fail in the development of their vaccines because of lack of knowledge on how to correctly formulate the adjuvants. This shortcoming led the World Health Organization to advocate for the establishment of the Vaccine Formulation Laboratory at the University of Lausanne, Switzerland. The primary mission of the laboratory is to transfer adjuvants and formulation technology free of intellectual property rights to academic institutions, small biotechnology companies and developing countries vaccine manufacturers. In this context, the transfer of an oil-in-water emulsion to Bio Farma, an Indonesian vaccine manufacturer, was initiated to increase domestic pandemic influenza vaccine production capacity as part of the national pandemic influenza preparedness plan.

Value of sTREM-1, procalcitonin and CRP as laboratory parameters for postmortem diagnosis of sepsis.

OBJECTIVES: Triggering receptor expressed on myeloid cells-1 (TREM-1) was reported to be up-regulated in various inflammatory diseases as well as in bacterial sepsis. Increased cell-surface TREM-1 expression was also shown to result in marked plasma elevation of the soluble form of this molecule (sTREM-1) in patients with bacterial infections. In this study, we investigated sTREM-1, procalcitonin and C-reactive protein in postmortem serum in a series of sepsis-related fatalities and control individuals who underwent medico-legal investigations. sTREM-1 was also measured in pericardial fluid and urine. METHODS: Two study groups were prospectively formed, a sepsis-related fatalities group and a control group. The sepsis-related fatalities group consisted of sixteen forensic autopsy cases. Eight of these had a documented clinical diagnosis of sepsis in vivo. The control group consisted of sixteen forensic autopsy cases with various causes of death. RESULTS: Postmortem serum sTREM-1 concentrations were higher in the sepsis group with a mean value of 173.6 pg/ml in septic cases and 79.2 pg/ml in control individuals. The cutoff value of 90 pg/ml provided the best sensitivity and specificity. Pericardial fluid sTREM-1 values were higher in the septic group, with a mean value of 296.7 pg/ml in septic cases and 100.9 pg/ml in control individuals. The cutoff value of 135 pg/ml provided the best sensitivity and specificity. Mean urine sTREM-1 concentration was 102.9 pg/ml in septic cases and 89.3 pg/ml in control individuals. CONCLUSIONS: Postmortem serum sTREM-1, individually considered, did not provide better sensitivity and specificity than procalcitonin in detecting sepsis. However, simultaneous assessment of procalcitonin and sTREM-1 in postmortem serum can be of help in clarifying contradictory postmortem findings. sTREM-1 determination in pericardial fluid can be an alternative to postmortem serum in those situations in which biochemical analyses are required and blood collected during autopsy proves insufficient.

Effect of collection and preprocessing methods on neutrophil elastase plasma concentrations.

OBJECTIVES: Elevated plasma levels of the elastase alpha 1-proteinase inhibitor complex (E-alpha 1 PI) have been proposed as a marker of bacterial infection and neutrophil activation. Liberation of elastase from neutrophils after collection of blood may cause falsely elevated results. Collection methods have not been validated for critically ill neonates and children. We evaluated the influence of preanalytical methods on E-alpha 1 PI results including the recommended collection into EDTA tubes. DESIGN AND METHODS: First, we compared varying acceleration speeds and centrifugation times. Centrifugation at 1550 g for 3 min resulted in reliable preparation of leukocyte free plasma. Second, we evaluated all collection tubes under consideration for absorption of E-alpha 1 PI. Finally, 12 sets of samples from healthy adults and 42 sets obtained from critically ill neonates and children were distributed into the various sampling tubes. Samples were centrifuged within 15 min of collection and analyzed with a new turbidimetric assay adapted to routine laboratory analyzers. RESULTS: One of the two tubes containing a plasma-cell separation gel absorbed 22.1% of the E-alpha 1 PI content. In the remaining tubes without absorption of E-alpha 1 PI no differences were observed for samples from healthy adult patients. However, in samples from critically ill neonates or children, significantly higher results were obtained for plain Li-heparin tubes (mean = 183 micrograms/L), EDTA tubes (mean = 93 micrograms/L), and citrate tubes (mean = 88.5 micrograms/L) than for the Li-hep tube with cell-plasma separation gel and no absorption of E-alpha 1 PI (mean = 62.4 micrograms/L, p < 0.01). CONCLUSION: Contrary to healthy adults, E-alpha 1 PI results in plasma samples from critically ill neonates and children depend on the type of collection tube.