high quantities in an antigen-specific manner following stimulation with antigens from a variety of pathogens, including CMV and MTB [4,5]. Such analyses have used Northern blotting, ELISA, Elispot, flow cytometry, and multiplex to measure IP10 and MIG concentration in patient serum or after in vitro stimulation with antigens. Subsequently, IP10 and MIG were proposed as adjunct biomarkers for tuberculosis (TB). However, measuring MIG and IP10 production with these techniques did not reach the sensitivity of the standard IFN-c Elispot [5,6,7,8]. Interestingly, a recent study in active tuberculosis has shown that measuring IP10 by Elisa in addition to IFN-c increases diagnostic sensitivity [9,10]. Real time qPCR has been used frequently to measure the antigen- specific immune responses of very small populations ofcells and is gaining popularity in vaccine immunology . A recent study used qPCR for MIG to detect responses in separated PBMCs to vaccines in a controlled setting  and has shown that MIG expression may correlate with protection against malaria . It is a versatile tool and can be used to measure the expression of virtually any mRNA transcript. We therefore sought to develop a novel qPCR-based assay for MIG and IP10 for the detectionofpathogen-specificTcells and apply this to the detectionof MTB infection, as an example of its application.
The primary aim of this study was to develop and test a nanoparticle-based assay for the sensitivedetectionof genomic bacterial biomarkers of an intracellular pathogen in clinical samples. Preliminary studies demonstrated that the hMRS can specifically detect a conserved genomic element (IS900) in MAP’s genome, and not in other microorganisms. Through additional studies, we identified that the sensitivity of the nanosensors was equal to one genome copy even in minimally processed samples, allowing detectionof MAP in clinical isolates, tissue homogenates and blood. Compared to nPCR, hMRS had improved perfor- mance and yielded faster results. Consequently, this translates to lower costs, since the magnetic nanosensors are fairly easy and cheap to manufacture in larger quantities, and the assay does not require multiple oligonucleotide primer pairs, expensive enzymes and nucleotides. Detection was performed using a simple table-top relaxometer that measured the increase in the water proton relaxation time (NMR signal) that occurred after the hMRS probes bound to their genomic target. The unprecedented detection limit of this technique is due to the build-in water relaxation amplification that results upon a single binding event. Figure 5. Detectionof Mycobacterium avium spp paratuberculosis ’ genomic marker in clinical samples with hMRS and direct nPCR. The hMRS detected MAP’s IS900 region in minimally processed blood samples (Cohort 1, n = 34). Correlation of the hMRS findings was achieved through pure DNA extraction from white blood cells and direct nPCR. Results are means 6 SE.
evaluated. Current methods of isolation and detectionof Phytophthora in oak roots, based mainly on baiting methods, however, are of relatively low effectiveness and reliability. More sensitive, molecular based diagnostic techniques are required. Indeed, the importance of Q. suber plantations to the Portuguese economy (Portugal ranks first in world cork production), and the social and environmental importance of cork oaks in the preservation of rural communities and in the ecological stability of the ecosystem on which they depend, further emphasizes the need to develop reliable, highly specific, sensitive and rapid methods to detect this pathogen. Several methods for identification of P. cinnamomi on roots of eucalyptus and avocado using hybridization techniques with DNA probes and }or PCR, have been developed previously (K. Sivasithamparam & P. O’Brien, pers. comm. ; Dobrowolski & O’Brien, 1993 ; Judelsen & Messenger-Routh, 1996). The targets for DNA amplification were the regions between the 18S and 5,8S (ITS I ; internal transcribed spacer region I) and between the 5,8S and 25S (ITS II) of ribosomal DNA. These regions exhibit useful interspecific variability (Lee & Taylor, 1992).
Two approaches were attempted for sensitivity assessment in this study. Firstly, the DNA concentration was measured spectrophotometrically and then diluted in different proportions in nuclease free water and tested for PCR amplification. The limit ofdetection for target DNA was 1 pg (Figure 3) and no amplification was obtained up to a dilution of 0.1 pg. Ot has been reported that the primers that amplify a short fragment of a target DNA give high sensitivity and the detection limit is enhanced to 1 pg (Amaral et al., 2014; Song et al., 2017). Secondly, the meat sample was adulterated with meat mixture consisting of other species in different proportions. The meats of different animal species were mixed in various combinations and proportions for this purpose. A total of seven combinations and eight proportions were included in the study and each of the combination and proportion was tested in triplicates. All the samples yielded specific amplification product suggesting that mixing the meat from different animal species did not affect the PCR amplification. The results of detecting the animal species from adulterated meat samples were presented such as duck meat in buffalo meat (Figure 4). Ot was found that species-specific PCR developed was highly sensitive to identify adulteration up to the extent of 0.1% (w/w). For all species, it was observed that the lower the percentage of the target meat in the admixture, the fainter the band obtained in the PCR with the corresponding specific primers. Similarly, a minimum detection limit of 0.1-0.01% (w/w) for meat products was found in various literatures (Karabasanavar et al., 2013; Ali et al., 2014; Amaral et al., 2015; Kim & Kim, 2017). Some other workers, especially by real-time PCR and other PCR methods, could even detected <0.01% (w/w) in the adulterated meat mixtures (Kesmen et al., 2012; Cho et al., 2014; Floren et al., 2015). However, the second approach is most preferred since it is closer to the actual conditions of meat adulteration in the market.
Phytoplasmas are non-helical obligate parasites that belong to the prokaryotic class Molli- cutes, and are transmitted by sap-feeding insects and vegetative plant propagation materials [15–18]. Diagnosis of phytoplasma pathogens has proven difficult because they cannot be cul- tured in vitro [19–21]. Before the advent ofmolecular techniques, disease symptoms, insect or dodder graft transmission to host plants, and electron microscopy were used for detectionof phytoplasmas . Following the initial cloning of phytoplasma DNA , nucleic acid-based applications have been developed for the detection and identification of phytoplasmas in plants and vectors. Polymerase chain reaction (PCR) amplification of 16S rRNA with either phyto- plasma species-specific primers or phytoplasma group-specific primers has been preferred widely for molecular diagnostics [20,24]. In particular, the nested-PCR approach has allowed the establishment of a specificdetectionof different species and strains of phytoplasmas . Until recently, 28 phytoplasma groups and 50 subgroups have been identified by PCR tech- niques . Although PCR methods have been used widely in phytoplasma identification and classification, they have many disadvantages, including time-consuming multi-step analyses with limited sensitivity, requirement for intense labor, and the risk of cross contamination dur- ing manipulation [27,28].
T cell response. Our initial observation that increasing doses ofT. cruzi caused an acceleration ofT. cruzi parasitemia without increasing mouse susceptibility provided a suitable experimental model to study this issue. As observed with T. cruzi infection, an inverse relationship was described Figure 5. Specific cytotoxicity in C57BL/6 mice challenged with irradiated or non-irradiated trypomastigotes ofT. cruzi. Groups of C57BL/6 mice were challenged or not i.p. with 10 3 or 10 4 irradiated or non-irradiated bloodstream trypomastigotes of the Y strain ofT. cruzi. A) Fifteen days after challenge, the in vivo cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of 4 mice6SD per group. B) Fifteen days after challenge, IFN-c producing spleen cellsspecific to the peptide VNHRFTLV were estimated by the ELISPOT assay. The results represent the mean number of SFC per 10 6 splenocytes6SD (n = 4). Asterisks denote statistically significant differences (P,0.05) when we compared mice challenged with irradiated or non-irradiated trypomastigotes ofT. cruzi . Results are representative of two independent experiments. doi:10.1371/journal.pone.0000393.g005
As a close cousin of M. tuberculosis, M. marinum is sensitive to most standard antibiotics used to treat tuberculosis, and also used to validate various screenings protocols [62,71]. In our host-pathogen infection model, most first line anti-tubercular drugs are efficient. Isoniazid (INH), ethionamide and ethambutol were active at 30 mM and efficiently blocked intracellular mycobacterial growth, only pyrazinamide was not active (Figure 2A). A rifamycin family derivate, rifabutin, was the most potent antibiotic in our infection assay with an MIC around 0.25 mM (Figure 2B). We also confirmed that rifabutin curing of an M. marinum infection occurred in a dose-dependent manner, and was consistent with confocal microscopy observations of infected cells (Figure 2B and C). At three days post infection, the intracellular bacteria load drastically diminished in presence of rifabutin, as reported by the total fluorescence intensity of bacteria inside infected cells (Figure 1). It is notable that rifabutin action on infection also restored host cell growth in a dose-dependent manner, as judged by the host cell density in Figure 2C. Finally, we assayed a panel of known specific anti-tubercular compounds and broad-spectrum antiobics to treat another intracellular pathogen infection, L. pneumophila, in the A. castellanii host (Figure 3A). Despite the common ability of these pathogens to avoid phago-lysosomal fusion, the Legionella-containing vacuole (LCV) differs from the M. marinum phagosome-derived compartment, and therefore, as expected, most of the anti-tubercular antibiotics have no effect on L. pneumophila replication. Only drugs such as floxacins  and rifampin , already reported to be active against L. pneumophila, cure A.castellanii infected cells.
Here we presented a simple and effective membrane mimetic microfluidic device with anti- body conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely- bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disinte- grate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.
In the present study, we evaluated three methods for detectionof residual undifferentiated hiPSCs in hiPSCs-derived differenti- ated cells: soft agar colony formation assay, flow cytometry and qRT-PCR. Table 1 summarizes the advantages and disadvantages of the assays associated with product tumorigenicity. The soft agar colony formation assay is known to be more sensitive in the detectionof certain types of tumorigenic cells, compared to in vivo methods using immunocompromised mice . However, we found the soft agar colony formation assay unsuitable for detecting residual undifferentiated hiPSCs, presumably attributable to the dissociation-induced apoptosis of hiPSCs . On the other hand, flow cytometry and qRT-PCR assays were found to be able to detect a trace amount of undifferentiated cells. These two assays have been exploited for characterization of stem cell-based products, as well as undifferentiated pluripotent stem cells, but the present study is the first analytical and quantitative approach designed to evaluate the detectionof residual undifferentiated cells in products derived from human pluripotent stem cells. The advantage of the flow cytometry assay is that it is able to identify undifferentiated cells. Unfortunately, the results are greatly affected by gating, and only the cells expressing the marker protein are detectable. On the other hand, the advantages of qRT- PCR are its rapidity, quantitativity and high sensitivity, whereas its disadvantage is that only the cells expressing the marker gene are detectable. Although the in vivo tumorigenicity assay is costly and time-consuming, it can directly analyze tumor formation in a specific microenvironment where the product is implanted (eg. retina). Therefore, a combination of relevant in vitro and in vivo assays would be necessary to ensure the safety of a product derived from human pluripotent stem cells. The rationale for the choice ofspecific assays would be justified, based on their characteristics shown in Table 1.
been optimized and clinically validated using appropriate positive and negative controls elsewhere . This assay can detect 18 of the most clinically relevant and common BCR-ABL mutations [14,29–30]. PCR amplifications were performed exactly as reported, without changing any of the reagents, PCR mix formulations and thermal profile . The sequences of ASO primers specific for each mutation with the corresponding annealing temperatures are given in Table 2. HL60 cell line (ATCC # CCL-240 TM ) was used as a negative control in ASO- PCR reactions. Although we used pre-validated ASO-PCR assays and reproduced those assays using exactly same reaction conditions, reagents and PCR mix formulation, to eliminate the possibility of false-positive results ASO-PCR products were sequenced on both strands using an automated ABI377 sequencer (Applied Biosystems). Sequences were analyzed with Sequence Analysis software V3.3 and Sequence Navigator software V1.0.1 (Applied Biosystems). A mutation was considered present only if it was detected in both strands in two or more independent ASO- PCR amplified products [14,29–31].
Due to the widely supported theory of bovine spongiform encephalopathy (BSE) spread in cattle by contaminated animal feeds, screening of feed products has become essential. For many years, manufacturers have used blood and plasma proteins as high quality ingredients of foods for both pets and farm animals. However, in Europe, the Commission Regulation 1234/2003/EC temporally bans the use of processed animal proteins, including blood-derivative products, in feedstuffs for all farm animals which are fattened or bred for the production of food. This regulation has some exceptions, such as the use of non ruminant blood products into the feed of farm fish. Authorization of the re-introduction of these proteins into animal feed formulations, especially non ruminant proteins into the feed for non ruminant farm animals, is expected when adequate control methods to discriminate ruminant proteins exist. Currently, the number of validated methods to differentiate the species of origin for most of the animal by-products is limited. Here we report the development of a rapid and sensitive polymerase chain reaction (PCR)-based assay, which allows detectionof bovine or porcine specific mitochondrial DNAfrom spray-dried blood derivate products (plasma, whole blood and red cells), as a marker for bovine contamination in porcine products. Sample extracts, suitable for PCR, were easily and quickly obtained with the commercial PrepMan TM Ultra reagent
pCT amplicon. Figure 3 summarizes the number of amplicons in the partial profiles. Sixteen samples were pCT negative but positive for two or more of the eight remaining loci; 15 of these Figure 2. Representative clinical sample 9-plex profiles. Approximately half the positive electropherograms showed all 9 amplicons with saturated (Panel A) or intermediate (Panel B) peak signals, suggesting high copy numbers of Ct. The remaining positive samples generated incomplete profiles, having fewer than 9 amplicons and suggesting low Ct numbers. For example, Panel C shows the pCT peak and three other amplicons, Panel D shows pCT and two other amplicons, and Panel E shows no pCT and two other amplicons in blue and red. Panel F is a negative clinical sample showing nonspecific peaks (circled in green) that are readily distinguished from Ct amplicons based on molecular weight and fluorescent dye color. The 353 bp peak in blue is readily distinguished from a true 349 bp pCT amplicon in blue. The 230 bp peak in red is readily distinguished from the 4 red-labeled specific amplicons, all of which differ in size from the artifact by at least 120 bp.
A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive and specificdetectionof African swine fever virus (ASFV). A set of LAMP primers was designed based on the sequence of the ASFV gene K205R. Reaction temperature and time were optimized to 64 o C and 60 min, respectively. LAMP products were detected by agarose gel electrophoresis or visually with the addition of fluorescent dye. The detection limit of the LAMP assay was approximately 6 copies of the target gene per microliter, 100 times more sensitive than conventional PCR. LAMP is a simple and inexpensive molecularassay format for ASFV detection. To date, African swine fever has not been reported in China. LAMP can be used to monitor ASFV spread into China, thereby reducing the threat of ASF.
detectable using the MHT, for which the sensitivity and specificity of the screening is known to be very good (1,2). However, the weakest aspect of the MHT is specificity. In a recent study using K. pneumoniae ATCC 700603 as the indicator strain instead of E. coli ATCC 25922 in a test procedure, the MHT had 97% sensitivity and 100% specificity (3). Another study reported a problem with sensitivity in expressing NDM-1 strains and found that specificity increased with the addition of ZnSO 4 (4).
Biodegradable polymers have increased the application of different polymer-based drug delivery systems (e.g. NPs, micelles and dendrimers) in oncology. Several kinds of polyme- Several kinds of polyme- ric based-NPS (e.g. albumin, poly(alkylcyanoacrylate), poly(lactic acid), poly(glycolic acid), poly(lactide-co-glycolide)) are tested for the treatment of various cancers (Miele et al.). Multi- (Miele et al.). Multi- functional polymeric micelles for targeting ligands and/or imaging and/or therapeutic agents are being actively developed (Nasongkla et al.). Dendrimers are versatile particles regarding their size and functionality. The easily modifiable surface characteristic of dendrimers enables them to be simultaneously conjugated with several molecules including therapeutic drugs, targeting ligands and/or other components to increase cancer specificity (Wang and Thanou).
A FlashQuant Workstation equipped with a 4000 QTRAP mass analyzer (MDS Analytical Technologies, Concord, Ontario, Canada) was used for automated measurements of the samples. The spots were measured in the selected reaction monitoring mode (unit resolution) by moving the target plate in a straight horizontal line at a speed of 1 mm per second while firing of the laser at 1,000 Hz, which corresponds to an analysis time of 4.5 seconds per spot. For specific setting see Table 1. Analyst 1.4.2. and FlashQuant 1.0 software (MDS Analytical Technologies, Concord, Ontario, Canada) were used to operate the instrument and for automated data analysis. A calibration curve was constructed by plotting the analyte-to-internal standard area ratios against the concentrations spiked using a 1/concentration 2 weighed linear curve. Quality controls were used to check the validity of the calibration curves. The absolute lower limit of quantification was set at the mean analyte area in the zero samples +10 times the standard deviation. Samples were spotted onto Opti- TOF 384 well stainless steel target plates (123681 mm; MDS Analytical Technologies, Concord, Ontario, Canada).
The combination of positron emission tomography (PET) with magnetic resonance imaging (MRI) has been the subject of several studies in recent years. Positron emission tomography is the most sensitive and specific imaging modality in the detectionof metabolic changes, but presents limited spatial resolution. On the other hand, MRI presents a significant spatial resolution, besides evaluating soft tissues signal intensity with excellent contrast resolution. The present iconographic essay is aimed at demonstrating the potential clinical application of PET/MRI coregistration. The studies were performed in a dedicated PET unit with 18
are observed in NMO patients  unless NMO-IgG and complement were co-injected directly into the brain in a traumatic approach . Furthermore, intravenous or intraperitoneal transfer of immunoglobulin fractions from NMO patients did not induce astrocytic damage in laboratory animals whose blood brain barrier was leaky in the absence of inflammatory stimuli  suggesting that lesion development in NMO may not exclusively rely on effector functions of NMO-IgG. In addition, the generation of anti-AQP4 antibodies of the IgG1 isotype in the peripheral immune compart- ment inevitably requires class-switch recombination in antigen specific B cells and thus cognate T cell help [24,25]. Therefore, we hypothesized that there must be an anti-AQP4 specificT cell response in NMO. In the present study, we tested the immunoge- nicity of AQP4 in C57BL/6 mice and identified the major I-A b restricted immunogenic determinants of AQP4. These data might build the foundation to develop animal models for NMO that include both antigen specificT cell and B cell mediated immunopathology.
Samples were inoculated onto Enterococcosel ™ agar (BBL, USA) supplemented with 6mg of vancomycin per mL and incubated at 35ºC for up to 72h. Suspicious colonies (Esculin positive) were planted onto sheep blood agar (BioMérieux, Brazil) and both resistance to vancomycin and identiication were conirmed using disk difusion and/or the Vitek system, following Clinical Laboratory Standards Institute criteria for deinition of resistance 9 .
Streptococcus _neumonia (pneumococcus) is an extracellular bacte- rium that causes significant mortality and morbidity globally . Young children are often nasally colonised and also have the highest incidence of pneumococcal infections. However with time, the rate of colonisation and infection falls and by late childhood the prevalence of nasal colonisation reaches a low-point – a state that persists into adulthood, although the incidence of pneumo- coccal infection increases in the elderly despite their maintaining relatively low rates of colonisation [2,3,4]. Pneumococcal exposure can lead to the generation of both B-cell and T-cell immune responses to polysaccharide and protein antigens [5,6,7], and although anti-capsular antibody responses generated by vaccina- tion in children can prevent subsequent colonisation, the natural acquisition of immunity to pneumococcus precedes detectable rises in anticapsular antibody responses . Furthermore, in adults the possession of high titre anti-capsular antibody responses does not necessarily protect against pneumococcal disease in selected patients . T-cells can play an important role in the development and maintenance of class switched antibody responses, although T-cell independent B cell class switching can also occur. Indeed, anti-pneumococcal protein antibody responses are T-cell depen- dant  and T-cell responses, as expected, are detectable in adults and children to both whole pneumococcus and pneumococcal