The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%).

Trypanosoma cruzi: metacyclogenesis in vitro - I. Changes in the properties of metacyclic trypomastigotes maintained in the laboratory by different methods

In this work we have studied the modifications in the biological properties of Trypanosoma cruzi when the parasite is maintained for a long time in axenic culture. The studies were done with a clone from an avirulent strain (Dm30L) and a non-cloned virulent strain (EP) of T. cruzi. Both parasiteswere maintained, for at least three years, by successive triatomine/mouse alternate passage (control condition), or by serial passage in axenic medium (culture condition), or only in the mouse (mouse condition). The comparison between parasites of culture and control condition showed that metacyclogenesis capacity was reduced in the former and that the resulting metacyclics displayed an attenuatedvirulence. In order to compare the virulence of metacyclics from the urine of the insect vector, Rhodnius prolixus were infected by artificial feeding with parasites of the control or culture condition. After three triatomine/triatomine passages, there was observed an almost identical biological behavior for these parasites, hence indicating that the maintenance of T. cruzi for a long time in axenic culture affects the differentiation capacity and the virulence of the parasite. Additionally, it was demonstrated that it is possible to maintain T. cruzi exclusively through passages in the invertebrate host.

Deicer Scaling Resistance of Concrete Mixtures Containing Slag Cement Phase 2: Evaluation of Different Laboratory Scaling Test Methods, TPF-5(100), 2012

With the use of supplementary cementing materials (SCMs) in concrete mixtures, salt scaling tests such as ASTM C672 have been found to be overly aggressive and do correlate well with field scaling performance. The reasons for this are thought to be because at high replacement levels, SCM mixtures can take longer to set and to develop their properties: neither of these factors is taken into account in the standard laboratory finishing and curing procedures. As a result, these variables were studied as well as a modified scaling test, based on the Quebec BNQ scaling test that had shown promise in other research. The experimental research focused on the evaluation of three scaling resistance tests, including the ASTM C672 test with normal curing as well as an accelerated curing regime used by VDOT for ASTM C1202 rapid chloride permeability tests and now included as an option in ASTM C1202. As well, several variations on the proposed draft ASTM WK9367 deicer scaling resistance test, based on the Quebec Ministry of Transportation BNQ test method, were evaluated for concretes containing varying amounts of slag cement. A total of 16 concrete mixtures were studied using both high alkali cement and low alkali cement, Grade 100 slag and Grade 120 slag with 0, 20, 35 and 50 percent slag replacement by mass of total cementing materials. Vinsol resin was used as the primary air entrainer and Micro Air® was used in two replicate mixes for comparison. Based on the results of this study, a draft alternative test method to ASTM C762 is proposed.

A study of combustion phenomena in circulating fluidized beds by developing and applying experimental and modelling methods for laboratory-scale reactors

The results shown in this thesis are based on selected publications of the 2000s decade. The work was carried out in several national and EC funded public research projects and in close cooperation with industrial partners. The main objective of the thesis was to study and quantify the most important phenomena of circulating fluidized bed combustors by developing and applying proper experimental and modelling methods using laboratory scale equipments. An understanding of the phenomena plays an essential role in the development of combustion and emission performance, and the availability and controls of CFB boilers. Experimental procedures to study fuel combustion behaviour under CFB conditions are presented in the thesis. Steady state and dynamic measurements under well controlled conditions were carried out to produce the data needed for the development of high efficiency, utility scale CFB technology. The importance of combustion control and furnace dynamics is emphasized when CFB boilers are scaled up with a once through steam cycle. Qualitative information on fuel combustion characteristics was obtained directly by comparing flue gas oxygen responses during the impulse change experiments with fuel feed. A one-dimensional, time dependent model was developed to analyse the measurement data Emission formation was studied combined with fuel combustion behaviour. Correlations were developed for NO, N2O, CO and char loading, as a function of temperature and oxygen concentration in the bed area. An online method to characterize char loading under CFB conditions was developed and validated with the pilot scale CFB tests. Finally, a new method to control air and fuel feeds in CFB combustion was introduced. The method is based on models and an analysis of the fluctuation of the flue gas oxygen concentration. The effect of high oxygen concentrations on fuel combustion behaviour was also studied to evaluate the potential of CFB boilers to apply oxygenfiring technology to CCS. In future studies, it will be necessary to go through the whole scale up chain from laboratory phenomena devices through pilot scale test rigs to large scale, commercial boilers in order to validate the applicability and scalability of the, results. This thesis shows the chain between the laboratory scale phenomena test rig (bench scale) and the CFB process test rig (pilot). CFB technology has been scaled up successfully from an industrial scale to a utility scale during the last decade. The work shown in the thesis, for its part, has supported the development by producing new detailed information on combustion under CFB conditions.

Inter-laboratory variation of in vitro cumulative gas production profiles of feeds using manual and automated methods

A study was conducted to estimate variation among laboratories and between manual and automated techniques of measuring pressure on the resulting gas production profiles (GPP). Eight feeds (molassed sugarbeet feed, grass silage, maize silage, soyabean hulls, maize gluten feed, whole crop wheat silage, wheat, glucose) were milled to pass a I mm screen and sent to three laboratories (ADAS Nutritional Sciences Research Unit, UK; Institute of Grassland and Environmental Research (IGER), UK; Wageningen University, The Netherlands). Each laboratory measured GPP over 144 h using standardised procedures with manual pressure transducers (MPT) and automated pressure systems (APS). The APS at ADAS used a pressure transducer and bottles in a shaking water bath, while the APS at Wageningen and IGER used a pressure sensor and bottles held in a stationary rack. Apparent dry matter degradability (ADDM) was estimated at the end of the incubation. GPP were fitted to a modified Michaelis-Menten model assuming a single phase of gas production, and GPP were described in terms of the asymptotic volume of gas produced (A), the time to half A (B), the time of maximum gas production rate (t(RM) (gas)) and maximum gas production rate (R-M (gas)). There were effects (P<0.001) of substrate on all parameters. However, MPT produced more (P<0.001) gas, but with longer (P<0.001) B and t(RM gas) (P<0.05) and lower (P<0.001) R-M gas compared to APS. There was no difference between apparatus in ADDM estimates. Interactions occurred between substrate and apparatus, substrate and laboratory, and laboratory and apparatus. However, when mean values for MPT were regressed from the individual laboratories, relationships were good (i.e., adjusted R-2 = 0.827 or higher). Good relationships were also observed with APS, although they were weaker than for MPT (i.e., adjusted R-2 = 0.723 or higher). The relationships between mean MPT and mean APS data were also good (i.e., adjusted R 2 = 0. 844 or higher). Data suggest that, although laboratory and method of measuring pressure are sources of variation in GPP estimation, it should be possible using appropriate mathematical models to standardise data among laboratories so that data from one laboratory could be extrapolated to others. This would allow development of a database of GPP data from many diverse feeds. (c) 2005 Published by Elsevier B.V.

Ice crystals growing from vapor in supercooled clouds between −2.5° and −22°C: testing current parameterization methods using laboratory data

The physical and empirical relationships used by microphysics schemes to control the rate at which vapor is transferred to ice crystals growing in supercooled clouds are compared with laboratory data to evaluate the realism of various model formulations. Ice crystal growth rates predicted from capacitance theory are compared with measurements from three independent laboratory studies. When the growth is diffusion- limited, the predicted growth rates are consistent with the measured values to within about 20% in 14 of the experiments analyzed, over the temperature range −2.5° to −22°C. Only two experiments showed significant disagreement with theory (growth rate overestimated by about 30%–40% at −3.7° and −10.6°C). Growth predictions using various ventilation factor parameterizations were also calculated and compared with supercooled wind tunnel data. It was found that neither of the standard parameterizations used for ventilation adequately described both needle and dendrite growth; however, by choosing habit-specific ventilation factors from previous numerical work it was possible to match the experimental data in both regimes. The relationships between crystal mass, capacitance, and fall velocity were investigated based on the laboratory data. It was found that for a given crystal size the capacitance was significantly overestimated by two of the microphysics schemes considered here, yet for a given crystal mass the growth rate was underestimated by those same schemes because of unrealistic mass/size assumptions. The fall speed for a given capacitance (controlling the residence time of a crystal in the supercooled layer relative to its effectiveness as a vapor sink, and the relative importance of ventilation effects) was found to be overpredicted by all the schemes in which fallout is permitted, implying that the modeled crystals reside for too short a time within the cloud layer and that the parameterized ventilation effect is too strong.

Comparison of two laboratory-developed PCR methods for the diagnosis of Pulmonary Tuberculosis in Brazilian patients with and without HIV infection

Abstract Background Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of pulmonary tuberculosis (PTB) is cheap and easy to use, but its low sensitivity is a major drawback, particularly in HIV seropositive patients. As such, new tools for laboratory diagnosis are urgently needed to improve the case detection rate, especially in regions with a high prevalence of TB and HIV. Objective To evaluate the performance of two in house PCR (Polymerase Chain Reaction): PCR dot-blot methodology (PCR dot-blot) and PCR agarose gel electrophoresis (PCR-AG) for the diagnosis of Pulmonary Tuberculosis (PTB) in HIV seropositive and HIV seronegative patients. Methods A prospective study was conducted (from May 2003 to May 2004) in a TB/HIV reference hospital. Sputum specimens from 277 PTB suspects were tested by Acid Fast Bacilli (AFB) smear, Culture and in house PCR assays (PCR dot-blot and PCR-AG) and their performances evaluated. Positive cultures combined with the definition of clinical pulmonary TB were employed as the gold standard. Results The overall prevalence of PTB was 46% (128/277); in HIV+, prevalence was 54.0% (40/74). The sensitivity and specificity of PCR dot-blot were 74% (CI 95%; 66.1%-81.2%) and 85% (CI 95%; 78.8%-90.3%); and of PCR-AG were 43% (CI 95%; 34.5%-51.6%) and 76% (CI 95%; 69.2%-82.8%), respectively. For HIV seropositive and HIV seronegative samples, sensitivities of PCR dot-blot (72% vs 75%; p = 0.46) and PCR-AG (42% vs 43%; p = 0.54) were similar. Among HIV seronegative patients and PTB suspects, ROC analysis presented the following values for the AFB smear (0.837), Culture (0.926), PCR dot-blot (0.801) and PCR-AG (0.599). In HIV seropositive patients, these area values were (0.713), (0.900), (0.789) and (0.595), respectively. Conclusion Results of this study demonstrate that the in house PCR dot blot may be an improvement for ruling out PTB diagnosis in PTB suspects assisted at hospitals with a high prevalence of TB/HIV.

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (â^ž), NLR (0.017), and Ef (99%).

Standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions

Adult honey bees are maintained in vitro in laboratory cages for a variety of purposes. For example, researchers may wish to perform experiments on honey bees caged individually or in groups to study aspects of parasitology, toxicology, or physiology under highly controlled conditions, or they may cage whole frames to obtain newly emerged workers of known age cohorts. Regardless of purpose, researchers must manage a number of variables, ranging from selection of study subjects (e.g. honey bee subspecies) to experimental environment (e.g. temperature and relative humidity). Although decisions made by researchers may not necessarily jeopardize the scientific rigour of an experiment, they may profoundly affect results, and may make comparisons with similar, but independent, studies difficult. Focusing primarily on workers, we provide recommendations for maintaining adults under in vitro laboratory conditions, whilst acknowledging gaps in our understanding that require further attention. We specifically describe how to properly obtain honey bees, and how to choose appropriate cages, incubator conditions, and food to obtain biologically relevant and comparable experimental results. Additionally, we provide broad recommendations for experimental design and statistical analyses of data that arises from experiments using caged honey bees. The ultimate goal of this, and of all COLOSS BEEBOOK papers, is not to stifle science with restrictions, but rather to provide researchers with the appropriate tools to generate comparable data that will build upon our current understanding of honey bees.

Handbook of methods for acid deposition studies : laboratory analyses for soil chemistry /

Mode of access: Internet.

Chemistry and food preparation, a laboratory manual for students of home economics; a series of experiments in elementary chemistry and experimental cookery designed for a scientific approach to the study of food preparation. Basic recipes and methods of combining ingredients are presented, together with variations that suggest the wide applicability of the fundamental principles.

Bibliography: p. 1

A study of matrix analysis methods for inelastic structures. Project no. 1467, Task no. 146701, prepared under Contract no. AF 33 (657)-8717 by Massachusetts Institute of Technology, Aeroelastic and Structures Research Laboratory.

Cover title.

Investigations of certain proposed methods of bank and embankment protection : part 1, full-scale experiments to determine method for protection of railroad embankments subject to overflow : part 2, laboratory investigations of the effectiveness of various proposed types of revetment.

Mode of access: Internet.

A Study of methods used in measurement and analysis of sediment loads in streams : report no.5: laboratory investigations of suspended sediment samplers /

Mode of access: Internet.