OPEN TEMPLATE of our PNEUMATIC & HYDRAULIC PRESSURE TESTING Method Statement including INSTRUCTIONS, that can be downloaded allowing editing and clean exporting for your project/company use.
Motor coordination was assessed with a rotating rod apparatus (Panlab Harvard Apparatus, Barcelona, Spain). The rod was 3 cm in diameter. The mice were placed on the rod when it was rotating at 4 rpm. The rotation speed was increased from 4 to 40 rpm within 5 min. The latency was recorded for each animal. This is the time (in seconds) before they fall. Each mouse was tested three times and the median time of the three trials was calculated. The results are expressed as the mean standard error of the mean (SEM) in each group.
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The mice were decapitated 6 hr after the acute PBS or LPS injection or 6 hr after the final PBS or LPS injection of the subacute treatment. The midbrains were dissected according to the method of Glowinski and Iversen [23] and stored at -80C until use. Total RNA was extracted using the QIAGEN RNeasy Lipid Tissue Mini kit (QIAGEN, Hilden, Germany). Real-time RT-PCR was carried out using QuantiTect SYBR Green RT-PCR system (QIAGEN). Primers for IL-1β, TNFα, TH and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were prepared by QuantiTect Primer Assays (QIAGEN). The amplification conditions were 40 cycles at 94C for 15 s, 55C for 30 s and 72C for 30 s. Quantitative data were obtained from the relative standard curve. mRNA expression was normalized using GAPDH as an endogenous control.
Blood samples were collected from 263 children admitted with fever or a history of fever to the pediatric clinic of the Agogo Presbyterian Hospital. The three different test methods PT, BN RDT and GM were performed independently by well trained and competent laboratory staff to assess the presence of malaria parasites. Results were analyzed and compared using GM as the reference standard.
In 107 (40.7%) of 263 study participants, Plasmodium sp. was detected by GM. PT and BN RDT showed positive results in 111 (42.2%) and 114 (43.4%), respectively. Compared to GM reference standard, the sensitivities of the PT and BN RDT were 100% (95% CI: 96.6-100) and 97.2% (95% CI: 92.0-99.4), respectively, specificities were 97.4% (95% CI: 93.6-99.3) and 93.6% (95% CI: 88.5-96.9), respectively. There was a strong agreement (kappa) between the applied test methods (GM vs PT: 0.97; p p
Half of the world's population is at risk of malaria, with an estimated 243 million cases worldwide out of which 85.6% cases occur in Africa [1]. The World Health Organization (WHO) estimated that 767 000 deaths occurred in Africa in 2008 of which almost 90% were children under five years [1]. Prompt parasitological confirmation by microscopy or alternatively by rapid diagnostic tests (RDTs) is recommended for all patients with suspected malaria before treatment is started [1]. This is to prevent the misuse of antimalarial drugs especially Artemisinin-based combination therapies thereby preventing the possible development of resistance in the parasite to these drugs. Treatment solely on the basis of clinical suspicion should be considered only when parasitological diagnosis is not accessible [1]. Thus, it is of concern that poor diagnostic standards such as the lack of skilled microscopists and inadequate or absence of quality control systems [2] continue to hinder effective malaria control.
Another major contributing factor is that the laboratory diagnosis of malaria has up to now relied nearly exclusively on light microscopy which is a valuable technique when performed correctly but unreliable and wasteful when poorly executed. A better utilization of microscopy and the development of alternative diagnostic techniques could substantially improve malaria control [3]. This study aimed at evaluating and comparing the novel Partec Rapid Malaria Test (PT) (Partec GmbH, Münster, Germany) and the recently established Binax Now Malaria Rapid Diagnostic Test (BN RDT) (Binax, Inc., Portland, ME, USA) in malaria diagnosis among children from an endemic area using Giemsa stain microscopy as the reference standard. In a first report we compared PT with GM in a separate collective of patients using a real time PCR assay as reference standard [4]. In this study we focused on the assessment of test result quality and applicability under the field conditions of a rural hospital laboratory.
Thick and thin blood films were prepared using standardized blood volumes of 10 μl and 2 μl, respectively. They were air-dried (thin film was fixed in absolute methanol), heat fixed and both stained with 10% Giemsa working solution for 12 minutes. A malaria blood film was considered negative after 100 high power fields (HPF) has been examined and no parasite observed. If parasites were observed, asexual malaria parasites were counted against 200 white blood cells (WBCs) on the thick film but all parasites in a final HPF were counted even if a count of 200 WBCs had been exceeded. The parasite count per microliter of blood was obtained by using the formula: (Parasite count/200WBC) Absolute WBC count [5]. If parasite density was more than 100 parasites/field in a thick film, the thin film was used for the count. Upon the observation of asexual malaria parasites, parasitized red blood cells (RBCs) were counted against 1,000 RBCs. The parasite count per microliter of blood was obtained by using the formula: (Parasite count/1000RBC) Absolute RBC count [6]. The thin film was used for species identification of detected malaria parasites. Absolute WBC and RBC counts were estimated by using the Sysmex KX 21N hematology auto analyzer (Sysmex Corporation, Kobe, Japan). In addition, gametocytes, schizonts and other blood parasites, when present, were reported to the clinicians. Known positive and negative samples were used as controls for freshly prepared Giemsa working solution each day.
Compared to the reference standard, the sensitivities of the PT and BN RDT were 100% (95% CI: 96.6-100) and 97.2% (95% CI: 92.0-99.4) respectively, and the specificities were 97.4% (95% CI: 93.6-99.3) and 93.6% (95% CI: 88.5-95.7) respectively (Table 3).
Table 3 shows the degree of agreement (kappa), with a 95% confidence interval, observed between the different test methods. Overall, there was a high degree of agreement between the test methods and the reference standard.
PT uses a fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) which detects intracellular double stranded DNA which is present within Plasmodium-infected erythrocytes. The bright shiny dots within infected erythrocytes under UV light is extremely characteristic for malaria and has a very high PPV in areas mainly endemic with P. falciparum [4]. One major limitation of PT is the disability of specific identification of Plasmodia and the differentiation of the species. Compared to GM, PT exhibited four false-positive results which have not been further investigated. PT could also be more sensitive than GM in detecting low number of parasites [4]. The presence of artifacts such as non-specific aggregated DAPI, immature erythrocytes or bacterial cells might have been misinterpreted as plasmodial DNA. The performance characteristics of the tests were very similar as indicated in their sensitivities, specificities, PPV and NPV with very good agreements to the GM reference standard (Table 3). Our study findings correlate well with the results of a study conducted in Sudan for adults using the Partec CyScope when compared only with conventional Giemsa stained microscopy [27] as well as with our first report comparing this malaria test in another patient's collective [4]. In this first study we could attest to the fact that the PT has a high sensitivity and specificity by referring to the highly sensitive gold standard RT-PCR which, however, cannot be applied in field studies in contrast to the PT assay [4]. Both studies [4, 27] confirmed that PT requires very little training and has a short turnaround time of averagely 5 minutes per test. In addition our findings regarding PT performance characteristics and applicability under field conditions were confirmed by a study conducted in Uganda [28]. This study also underlines the disability of specific identification and differentiation of Plasmodia species as the disadvantage of PT. However, the characteristic fluorescence of infected erythrocytes has a very high PPV in areas mainly endemic with P. falciparum.
The accepted level of sensitivity for a rapid diagnostic test in diagnosing malaria is a sensitivity of 95% [31]. Compared to the reference standard, both methods were sensitive and specific enough to be used as diagnostic tools for the diagnosis of malaria in endemic areas.
The quantification of the CytB G143A mutation in Bgt using ASqPCR was first conducted by Fraaije et al.26 using the intercalating dye SYBR 1, with the assay detecting as low as 1 in 10,000 copies of the mutant A143 allele in a sample extraction that contained a mix of fungal and wheat DNA. A qPCR assay developed for the analysis of mutation Y136F in the Cyp51 target site of E. necator had a limit of detection and a limit of quantification of 0.85 and 2.85%, respectively44. More recently, Zulak et al.43 developed a chip dPCR assay able to quantify mutations S509T and Y136F in the Cyp51 of B. graminis f. sp. hordei infected samples down to 0.2%. In our study, the quantification limit of the field-based G143A ASqPCR assay, when using a DNA extract containing a mixture of both A143 and G143 alleles, and wheat DNA, was 1.67% (Table 4). However, in samples with homogenous (homoplasmic) genotypes, in a background of wheat DNA, the assay successfully detected seven copies of the G143 or A143 alleles per reaction (Fig. 2b). The latter could be considered as the equivalent to a single spore detection, as each spore has multiple mitochondria and each mitochondrion may have multiple copies of the cytb gene within it36. A laboratory based G143A chip dPCR assay was also developed for comparative purposes. The G143A dPCR assay successfully quantified the cytb A143 allele in a background of G143 and wheat DNA down to a level of 0.33%, whereas its detection limit was estimated at 0.07% (Fig. 2a; Table 4). In general, dPCR has lower target detection limits and higher reproducibility, than qPCR, the latter due to the use of absolute quantification rather than relying on standard curves43,45,46. Due to these properties, dPCR could be considered the gold-standard in molecular detection of fungicide resistant populations. 2ff7e9595c
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