can be either direct demonstration or indirect demonstration of Leptospira from clinical specimens.
1.1.8 (a) Microscopic observation
Direct observation of Leptospira from body fluid or artificial EMJH culture can be carried out using a dark field microscope. Under the microscope, the leptospires appear as thin, bright and motile spirochetes. In order to be detectable in the microscopic field, the initial Leptospira concentration in clinical specimens should be 10 4 cells per mL (Toyokawa et al., 2011). A false negativity may happen when a low number of Leptospira present in the sample. In addition, this method is prone to false positivity when artefacts such as fibrin or protein threads and cellular debris are mistakenly identified as leptospires. Differential centrifugation can be performed to concentrate the organism, the technique is only useful for a large volume of specimens (WHO, 2013). Alternatively, some staining procedures, such as Warthin-Starry silver nitrate staining, immunohistochemistry and in situ hybridization to increase sensitivity and specificity. These methods, however, requires highly experienced technicians and laborious, hence are less useful as routine diagnostic tests (Ahmad et al., 2005).
Oesophagoscopy is considered the most sensitive and specific diagnostic technique for the detection of oesophageal spirocercosis , but is limited by high cost, availability of specialized equipment, requirement for general anesthesia, potential complications (e.g. oesophageal bleeding or rupture) and the need for clin- ical expertise for the accurate identification of S. lupi nodules [1, 5]. Although in the present study, up to 61% of the spirocercosis cases were detected as positive by the ITS1 HRM qPCR, seven cases verified by oesopha- goscopy were not detected as positive by any other methodology. This might be explained by the intermit- tent egg shedding into the feces, and low fecal egg num- bers, which might have been absent in the small volume of fecal sample obtained for DNA extraction, or single- sex worm oesophageal infection, resulting in the absence of egg shedding . Thus, we recommend analyzing a second fecal sample, obtained a few days after a negative result, since this can increase the positivity of detection in fecal samples, as previously suggested [21, 22].
Methods: The qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples. Results: The relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a lowqPCRvolume made the developed assay an economical alternative for the GAS detection. Conclusion: We showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.
contrast to the CellSearch system, in which images of cells are determined to be CTCs by software and need to be manually confirmed by an operator. This step can introduce subjectivity in scoring, which is significant when as few as one or two cells in a sample are sufficient to class the patient as ‘CTC-positive’. Recently, Kraan et al (2011) observed that image interpretation was the main contributor to between-laboratory variation. In addition, our assay is considerably less expensive, costing less than US$25 per sample, compared with approximately US$600 per sample for the CellSearch system (Kaiser, 2010). Finally, there already exists an equipment for the automation of the enrichment, cDNA preparation and QPCR steps in our assay, and hence it could potentially require little manual work or technical knowledge. Although this is a preliminary study and further validation involving additional patients would be required to confirm that an automated system could provide equivalent prognostic data, there is a potential for the use of such system in a clinical setting. The threshold for QDA positivity was set at the highest QDA value in the healthy controls group, resulting in 100% specificity, as previously described (Bosma et al, 2002; Weigelt et al, 2003). Although our assay’s sensitivity could have been augmented by lowering the threshold, priority was given to avoid false positives. In total, 81% of the advanced patients assayed were CTC positive vs 0% of the healthy controls (Figure 1). We hypothesised that enriching a sample for tumour cells before assessing tumour marker gene expression would be beneficial for assay sensitivity, and this appears to be the case. We had previously demonstrated that when tumour cell enrichment was not performed, 30% of advanced patients assayed from a similarly selected patient group had a positive QDA score indicating CTC positivity vs 0% of healthy controls (Weigelt et al, 2003). We also had previously shown that using a positive enrichment strategy for cells expressing both EpCam and ErbB2 antigens resulted in the detection of higher levels of tumour marker gene expression than enriching for cells expressing just one of the antigens (Molloy et al, 2008). Also, the multimarker gene expression panel used has obvious benefits over the use of a single marker for the detection of tumour cells. Finally, the use of the quadratic discriminant score function is not prone to subjectivity in scoring or inaccuracies in quantitation as immunohistochemical staining or densitometry of an electrophoresed nucleic acid band can be.
This study did not aim to evaluate sensitivity of molecular methods against standard T. cristatus survey methodologies. Egg searches were used to detect false negatives produced by qPCR and metabar- coding and in doing so, revealed some interesting results. Biggs et al. (2015) previously demonstrated qPCR had higher detection rate than egg searches (as well as torchlight, netting, and bottle trapping), but here we show this also holds true for metabarcoding. Importantly, absence of eggs does not infer absence of adults, and this method is highly dependent on weather conditions and water clarity (Biggs et al., 2015; Rees, Bishop, et al., 2014). Despite considerably higher detection rate of both eDNA approaches, eggs were recorded in a small number of ponds that were eDNA negative. eDNA analysis can incorrectly infer absence or low abundance of species if inhibi- tion or interference from non-target DNA has occurred (Goldberg et al., 2016). Alternatively, eDNA false negatives may have been a by- product of sampling strategy and effort for T. cristatus. Larger water volumes and/or more biological replication instead of pseu- doreplication (established T. cristatus eDNA sampling strategy) may improve detection (Andruszkiewicz et al., 2017; Bálint et al., 2017; Lopes et al., 2016). All methods revealed T. cristatus in ponds where other approaches failed, emphasising that these species monitor- ing tools are complementary and should be used in combination to achieve maximum detection probability. However, integrative strat- egies combining molecular and conventional tools are often not cost- efficient for most applications.
The fact that the SPUD amplicon stably amplifies at a significantly lower efficiency than does the SPUD amplicon-containing plasmid, we feel, shines light on a large misconception in qPCR. It is often assumed that the same target sequence, no matter how it is presented in the qPCR, should amplify with the same efficiency. We have never found this to be true in our work. E.g. when we compared endogenous sheep lung VEGF RNA splice variant targets to the same targets contained in plasmids (using both plasmids and sample at non-inhibitory dilutions; as established by PREXCEL-Q), the same target demonstrated a different efficiency of amplification. Since inhibition had been eliminated from these assays, there must be different geometries at work by which the same target, presented to qPCR in different contexts, will amplify at different efficiencies accordingly (J.M. Gallup, A. Van Geelen, unpublished). The differing efficiencies in such cases are thus not due to one target reaction (i.e. for the 101 bp SPUD amplicon) being inhibited while the other (SPUD plasmid) is not, rather, the geometry of target:primer-probe interaction (at the chosen thermocycling conditions) is most likely more optimal for the SPUD plasmid reaction that it is for the SPUD amplicon reaction. That is to say, at the conditions chosen, one reaction’s template context is more kinetically-conducive to efficient qPCR than the other, even though the same target is being amplified in both cases. It could be that the SPUD target, when held within the more thermodynamically stable context of a plasmid, is more readily amplified than is the SPUD amplicon itself.
After a literature review on unbound granular materials mechanical behaviour and on lowvolume roads pavement design methods, Chapters 4 and 5 discuss full scale trials carried out in Scotland on typical forest roads. The overall goal of the trials carried out within the Roads Under Timber Transport project was to establish the effect of weather and seasonal effects on the rutting of forest roads and to improve their performance while enabling the roads to be economically constructed and maintained. It appears that most of the rutting occurring in the sites surveyed came shortly after their construction/resurfacing, leading to the assumption that workmanship may be a highly important variable. Lack of compaction of the layer could be one of the likely reasons for the high initial rutting rates. Establishing the effect of weather on rutting further to the existing knowledge was, however, difficult to achieve; this was mainly due to the difficulties faced in monitoring traffic conditions. A newly developed method was needed to quantify permanent deformation development due to wandering traffic on a non-level pavement; this was achieved by the use of wheel path areas, and seemed to be a way forward in the analysis of rutting in unsealed roads.
Background: Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices.
This approach requires significantly more hands-on time, has a greater risk of contamination and makes multiplexing analysis more difficult if products are similar in size, compared to real-time PCR. Probe-based qPCR can overcome these issues. Additionally, specificity can be increased, and it allows for continuous monitoring of the PCR. The 18S rRNA gene (18S rDNA) is a highly conserved gene across all Leishmania species, despite having diverged from other similarly related species during the period Paleogene or Paleocene . The gene exists in between 50–200 copies per Leishmania genome, making it an excellent choice for a pan-Leishmania detection assay . To assess whether this target can be used in a novel diagnostic assay, based on bisulphite conversion and real-time PCR technologies, a series of experiments were performed to assess the limit of detection and sensitivity of the assay, and the new assay was compared to a cPCR, based on the ITS1 region, developed by Schönian et al. . The development of this novel bisulphite-converted, qPCR assay methodology, based on genus-specific primer and probe designs for the 18S rDNA, and its validation, is described in this paper. Furthermore, the bisulphite conversion and purification of protozoan DNA are discussed. The assay’s limit of detection was 10 cellular or genomic copies/PCR, with clinical sensitivity and specificity demonstrated to be 97.0% and 100%, respectively. The assay takes under 2.5 hours to complete, making the assay a potential diagnostic tool for both diagnostic and research laboratories worldwide.
The aims of this study were to evaluate two methods, qPCR and a chemiluminescent assay (ColiLight II), for rapid de- tection of E. coli in water, and to examine the survival and persistence of clinical E. coli in drinking water and biofilm using qPCR and ColiLight II. qPCR and ColiLight II were compared with a cultivation-based method (MPN), and sur- vival and persistence of four clinical E. coli strains in water and biofilms on stainless steel (SS) and polyethylene (PE) surfaces were studied in a flow-through reactor with non-disinfected drinking water using ColiLight II, qPCR, ATP bioluminescence, and MPN. ColiLight II and qPCR correlated well with MPN. In drinking water, some clinical E. coli strains showed prolonged survival in drinking water flow-through systems, and persisted 3 - 3.4 times longer than the theoretical washout due to incorporation into biofilms. Strain specific attributes can significantly affect detection and persistence of E. coli in drinking water matrices.
The ABI 7900HT qPCR machine does both the thermal cycling and fluorescence detection and also the measurements. The machine is connected to a computer with SDS software. It uses the ROX dye fluorescence to normalise the PCR product-related fluorescence signals. A baseline for the qPCR reaction is calculated based on the fluorescence acquired from the PCR products and it is reckoned the noise level of the early cycles when there is not yet detectable fluorescence from the amplification products (Figure 4) (QIAGEN - Data analysis 2013). The software produces a sigmoidal-shaped amplification plot (when using a linear scale, Figure 4), where the amount of detected fluorescence is plotted against the cycle number. The threshold is set to value above the baseline, but in the log-linear range of the plot. The threshold cycle (C T ) values are determined using this threshold: a C T value indicates the reaction cycle at which the amplification plot crosses the set threshold (Figure 4) (QIAGEN - Data analysis 2013). The higher the C T value, the lower the starting amount of the template: in Figure 4, sample A contains more starting template than sample B. C T values above 36 cycles are generally considered poorly reproducible and thus unreliable for e.g. parasite detection (ten Hove et al., 2007). An increase of approximately 3,5 cycles indicates a 10-fold decrease in the amount of the template in the original sample.
SUN, D., WANG, J., WU, R., WANG, C., HE, X., ZHENG, J. & YANG, H. (2010) Development of a novel LAMP diagnostic method for visible detection of swine Pasteurella multocida. Veterinary Research Communications 34, 649-657 TOWNSEND, K. M., BOYCE, J. D., CHUNG, J. Y., FROST, A. J. & ADLER, B. (2001) Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 39, 924-929
To understand the dynamics of virus infection, it is useful to elucidate the pathogenesis and persistence of viruses, allowing prediction of disease progression, and evaluating the effects of antiviral therapy as well (Iwami et al., 2013). In general, virus isolation techniques or polymerase chain reaction (PCR) along with mathematical models are useful to determine certain aspects of in-vitro virus infection that are usually associated with disease severity, such as: sites of infection, used for detection of Zika virus (ZIKV) in amniotic fluids of microcephaly fetuses (Calvet et al., 2016), target cells, for identification of alveolar macrophages and dendritic cells such as Measles virus (MV) primary target, when it is transmitted by aerosol inhalation in non-human primates (Lemon et al., 2011), and viral gene functions, such as in the Human immunodeficiency virus (HIV) Vif gene, which is capable of inhibiting APOβEC class restriction factors expression, ensuring viral replication. (Hultquist et al., 2011)
In addition to BioGX Inc, there are other companies, such as GE Life Sciences (Pittsburgh, PA, USA) and Cepheid (Sunnyvale, CA, USA) that can custom manufacture lyophilized PCR assays. It is also possible to custom-make and optimize lyophilized PCR assays in the laboratory as well. However, this can be a long, tedious and complex process. For example, in an attempt to lyophilize an internal positive control RNA to help ensure the accuracy of the detection of avian influenza virus RNA by reverse transcription (RT)-PCR and real-time RT-PCR, Das et al.  were forced to lyophilize without some of the enzymes because of stability concerns. Klatser et al. describes a complex procedure for developing of a freeze-dried, lyophilized PCR assay . In 2010, Cheung et al .  attempted to custom-make lyophilized assay for detection of influenza. However, the lyophilized reagents and the assay condition needed further optimization to improve the sensitivity and amplification efficiency. However, when a PubMed search was done in January 2014, there was no indication such optimization had been published more than three years later. Having a company custom-manufacture lyophilized assay(s) is likely to result in a more optimized, reproducible product, much more than attempting to custom-make lyophilized assay in a laboratory due to complexity of the procedure. However, the lyophilization process is likely to increase the cost of qPCR assay which is one of the main concerns of molecular assays. At the current rate, “wet” assay cost less than one dollar per reaction whereas lyophilized assay is more than ten dollars per reaction, increasing the cost of qPCR per reaction by more than ten-fold. It is therefore critical that the benefits of lyophi- lized molecular assays are assessed in the context of all the benefits gained with lyophilization and the cost savings that come along with it, such as reduced cost in transporting of reagents and no need for cold-chain.
nars have been conducted across the U.S., in Mexico, Canada and even Europe. Rain Bird has talked to more than 1,000 irrigation professionals about landscape low-volume irrigation. In the past, the most frequently asked question was, “Why should I use low-volume irrigation?” Today, however, most seminar attendees share the basic understanding that, when properly used, landscape low-volume irrigation products can save significant amounts of water. These products can also help keep water off walls, windows, sidewalks and streets. At the same time, the use of these products promotes healthier plant growth because water is delivered more slowly and at lower pressures at or near plant root zones.
Although it is regarded as a simple, robust, low cost and sensitive method in gene detection, qPCR often still produce compromising results, such as generating false positive and false negative results [Niwa et al., 2007]. These false results could be due to various factors, one of which is the lack of adequate quality controls. Positive control is one of the quality controls used in qPCR method to assure the optimum PCR reaction and prevent the false negative results caused by inhibitor in the reaction, the lack of DNA polymerase activity, and the error of thermal cycler [Hoorfar et al., 2004, Majidzadeh et al., 2014]. Therefore, the pre- sence of positive control is important in gene detection using qPCR method.
Rapid and specific detection of extended‑spectrum β‑lactamase‑producing (ESBL) bacteria is crucial both for timely antibiotic therapy when treating infected patients as well as for appropriate infection control measures aimed at curbing the spread of ESBL‑producing isolates. Whereas a variety of phenotypic methods are currently available for ESBL detection, they remain time consuming and sometimes difficult to interpret while being also affected by a lack of sensitivity and specificity. Considering the longer turnaround time (TAT) of susceptibility testing and culture results, DNA‑based ESBL identification would be a valuable surrogate for phenotypic‑based methods. Putative ESBL‑positive Enterobacteriaceae isolates (n = 330) from clinical specimen were prospectively collected in Bulgaria, Romania and Democratic Republic of Congo and tested in this study. All isolates were assessed for ESBL‑production by the E‑test method and those giving undetermined ESBL status were re‑tested using the combination disk test. A genotypic assay successively combining qPCRdetection of blaCTX‑M, blaTEM and blaSHV genes with a multiplex pyrosequenc‑ ing of blaTEM and blaSHV genes was developed in order to detect the most common ESBL‑associated TEM and SHV single nucleotides polymorphisms, irrespective of their plasmid and/or chromosomal location. This assay was applied on all Enterobacteriaceae isolates (n = 330). Phenotypic and genotypic results matched in 324/330 (98.2%). Accord‑ ingly, real‑time PCR combined with multiplex pyrosequencing appears to be a reliable and easy‑to‑perform assay with high‑throughput identification and fast TAT (~5 h).
The use of geosynthetics is widespread in the field of civil engineering. One particular application that has seen a great deal of use has been as a separating layer between the aggregate base layer and subgrade of lowvolume roadways. Since geosynthetics may be very stiff, they may provide an additional benefit of reinforcement to the roadway. For this study, only the reinforcement functions of geosynthetics were investigated. Long term separation and increased bearing capacity during construction were not considered. The research described herein was originated to investigate the reinforcement function of geosynthetics for typical Minnesota low-volume roadways. To this end, a series of numerical experiments were conducted using the finite difference program FLAC (1993). The tests consisted of a static, circular, 9 kip loading over a variety of typical surfaced and unsurfaced road cross sections that were reinforced with geotextiles and geogrids. The results are shown in terms of percent normalized deflection reduction and percent normalized accumulated standard axle load to a serviceability level of 2.5 (ASAL2.5) increase. Additionally, the effect of a geosynthetic reinforcement layer on the horizontal stress distribution is illustrated. The results of the study indicate that the addition of a geosynthetic does provide reinforcement to the roadway as long as the geosynthetic is stiffer than the subgrade material. However, for most of the cases studied, the benefit in terms of deflection reduction, was very small. Only for the poorest quality subgrades was the reinforcement benefit substantial.
The breast like any other human organ is a complex structure that is difficult to define as a specific geometric form. To calculate the volume, the three corresponding dimensions from the ROI of the CC and MLO views are needed. The two ROI images obtained from the above methods are two dimensional (2D) images but have two different perspectives. From the ROI image of the MLO view the height (hMLO) and the width (wMLO) can be obtained. Similarly, from the CC view of the ROI image the height (hCC) and the width (wCC) can be derived. The height in the MLO and CC views are of same value, so either of the ROI image to calculate the height can be considered. The width in CC and MLO views are the remaining two dimensions for calculating the volume of breast. The width in the MLO view can be defined as the region that extends from the point where the lateral thoracic artery enters the breast region to the point where skin crosses the pectoralis major, i.e. the infra-mammary fold. This fold is formed by the infra-mammary ridge, which is a band of fatty tissue formed by the folding over of the breast upon itself and is deeply connected to the sagging of breast in older women. To measure the extent of the breast the line adjoining these two points starting from the thoracic artery defined as point A to infra-mammary-fold defined as point B must be taken into consideration. A line AB has been drawn that forms the axis of the breast or wMLO. This is shown in figure 2.
A further evaluation of clinical serum detection was carried out to compare the novel developed method and ELFA. Linear regression analyses revealed good correlations between the novel developed method and the approved com- mercial kit. This indicated no significant statistical difference between the two methods and thus the newly established assay might be used for the clinical determination of PCT in human serum. Furthermore, the comparison of assay charac- teristics with the reported methods is shown in Table S1.