Immunomagnetic Separation Technique Research Articles

FEN1 upregulation mediated by SUMO2 via antagonizing proteasomal degradation promotes hepatocellular carcinoma stemness

PurposeMetastasis of hepatocellular carcinoma (HCC) critically impacts the survival prognosis of patients, with the pivotal role of hepatocellular carcinoma stem cells in initiating invasive metastatic behaviors. The Flap Endonuclease 1 (FEN1) is delineated as a metallonuclease, quintessential for myriad cellular processes including DNA replication, DNA synthesis, DNA damage rectification, Okazaki fragment maturation, baseexcision repair, and the preservation of genomic stability. Furthermore, it has been recognized as an oncogene in a diverse range of malignancies. Our antecedent research has highlighted a pronounced overexpression of protein FEN1 in hepatocellular carcinoma, where it amplifies the invasiveness and metastatic potential of liver cancer cells. However, its precise role in liver cancer stem cells (LCSCs) remains an enigma and requires further investigation. MethodsTo rigorously evaluate the stemness attributes of LCSCs, we employed sphere formation assays and flow cytometric evaluations. Both CD133+ and CD133- cell populations were discerningly isolated utilizing immunomagnetic bead separation techniques. The expression levels of pertinent genes were assayed via real-time quantitative PCR (RT-qPCR) and western blot analyses, while the expression profiles in hepatocellular carcinoma tissues were gauged using immunohistochemistry. Subsequent immunoprecipitation, in conjunction with mass spectrometry, ascertained the concurrent binding of proteins FEN1 and Small ubiquitin-related modifier 2 (SUMO2) in HCC cells. Lastly, the impact of SUMO2 on proteasomal degradation pathway of FEN1 was validated by supplementing MG132. ResultsOur empirical findings substantiate that protein FEN1 is profusely expressed in spheroids and CD133+ cells. In vitro investigations demonstrate that the upregulation of protein FEN1 unequivocally augments the stemness of LCSCs. In a congruent in vivo context, elevation of FEN1 noticeably enhances the tumorigenic potential of LCSCs. Conversely, inhibiting protein FEN1 resulted in a marked reduction in LCSC stemness. From a mechanistic perspective, there exists a salient positive correlation between the protein expression of FEN1 and SUMO2 in liver cancer tissues. Furthermore, the level of SUMO2-mediated modification of FEN1 is pronouncedly elevated in LCSCs. Interestingly, SUMO2 has the ability to bind to FEN1, leading to a inhibition in the proteasomal degradation pathway of FEN1 and an enhancement in its protein expression. However, it is noteworthy that this interaction does not affect the mRNA level of FEN1. ConclusionIn summation, our research elucidates that protein FEN1 is an effector in augmenting the stemness of LCSCs. Consequently, strategic attenuation of protein FEN1 might proffer a pioneering approach for the efficacious elimination of LCSCs.

Magnetic capture device for large volume sample analysis.

Immunomagnetic separation (IMS) techniques employing superparamagnetic particles can successfully isolate various components from mixtures. However, their utility can be limited for large-volume samples, viscous samples, or those containing a high density of particulate matter because of the need to generate high field gradients for particle recovery. Therefore, a new class of immunomagnetic particles was devised utilizing a single, macroscopic Pyrex spinbar conjugated with biorecognition elements to address these limitations. Advantages include an inherent capacity for effective mixing, an almost instantaneous recovery of the spinbar that can be performed without expensive equipment and with no loss of magnetic particles during processing, and reduced transfer of sample matrix. As a result, spinbars can provide an effective means for IMS with large-volume assays composed of complex matrices.

Optical Spectroscopy Based Microfluidic Platform for Detecting Pathogens Using Immunomagnetic Separation

Rapid detection of pathogens is crucial for controlling pathogenic diseases and improving the quality of food industry. This paper presents a microfluidic platform integrated with optical detection module to rapidly detect Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The detection module comprises a microfluidic chip embedded with fiber optics connected to photosensors and a laser source. Initially, the immunomagnetic separation technique was applied to isolate specific pathogens out of testing sample using magnetic particles coated with antibodies. The separated sample containing magnetic beads was loaded into the chip and passed by the monochromatic light in the detection module. The scattered light signals from the passing magnetic beads were collected by the photosensors coupled with fiber optics. The acquired raw data were pre-processed by removing noise and distortions for further analysis. A new calibration model known as the Optical Transformer (OptiTr) structure was used for classification of acquired data. Convolution-based classical model (ResNeXt) was used as baseline comparison. The system provided higher classification results using the OptiTr model for classifying P. aeruginosa and E. coli, with accuracy values of 99.57% and 94.59%, respectively. The blank samples without P. aeruginosa and E. coli showed a classification accuracy of 94.73%. The microfluidic platform has the capability to identify P. aeruginosa and E. coli with a detection limit of 101 CFU/mL. The technique does not require complex sample preparation methods or complicated laboratory tools. The developed technique has a 15-minute detection time with a 40-minute sample preparation duration.

Sensitive Molecular Detection for Salmonella Enterica Serovars Typhimurium

Introduction: Salmonella infections contribute significantly to gastroenteritis cases, with the National Salmonella Reference Laboratory reporting 500 isolates in 2022. However, traditional culture-based methods for detecting Salmonella in samples can take 4 to 7 days to confirm a positive result, which poses health risks due to delayed detection. Given these health risks, swift and accurate detection methods are essential to minimize both false-positive and false-negative outcomes. Salmonella, a gram-negative bacterium within the Enterobacteriaceae family, demonstrates remarkable hardiness, surviving for several weeks in dry environments and months in water. Although most serotypes of Salmonella cause relatively mild gastroenteritis, some, particularly those transmitted from animals to humans, can lead to severe, life-threatening conditions
 Methods: The qRT-PCR procedure involved the design of primers and probes targeting the same genes as the mPCR assay. These primer sets were reconfigured to generate smaller amplicons suitable for qRT-PCR systems
 Results: qRT-PCR process, TaqMan probes were meticulously designed for specific target genes: FAM dye was employed to detect STM2745, Cy5 dye was used for STM4492, and Rox dye was utilized to detect. A standard curve was constructed using Typhimurium LT2 genomic DNA. Each sample underwent duplicate analysis, and Rotor-Gene software was employed to assign threshold values for each channel.
 Conclusion: The effectiveness of our qPCR assay for the detection of Salmonella across a diverse array of matrices. Notably, our results unveiled distinct limits of detection for Salmonella in various samples. Specifically, a parallel vein, the deployment of a PCR assay, leveraging an immunomagnetic separation technique for DNA extraction, was studied by another group. While subsequent analysis of Salmonella detected via our assay may necessitate the full ISO SMT method for live culture isolation, this supplementary step can be seamlessly conducted alongside qRT-PCR.

A light-activated magnetic bead strategy utilized in spatio-temporal controllable exosomes isolation.

Tumor-derived exosomes are considered as a key biomarker in the field of liquid biopsy. However, conventional separation techniques such as ultracentrifugation, co-precipitation and column chromatography cannot isolate samples with high throughput, while traditional immunomagnetic separation techniques, due to steric effect of magnetic beads, reducing sensitivity of exosomes optical detection. Herein, we provide a novel and simple nanoplatform for spatiotemporally controlling extraction and elution of exosomes via magnetic separation and light-activated cargo release. In this system, magnetic beads are co-modified by photoresponsive groups -nitrobenzyl group and aptamers that are compatible with CD63-a highly expressed exosomal surface-specific protein. Through exosomes extracted from cell model and nude mice xenograft tumor model morphological characterization and proteomic analysis, results showed that our novel magnetic bead system outperformed current ultracentrifugation in serum exosome extraction in terms of extraction time, yield, and proportion of populations with high CD63 expression. This strategy may be a powerful tool for exosome isolation in clinical liquid biopsies of cancer disease.

Quantitative detection of trace VBNC Cronobacter sakazakii by immunomagnetic separation in combination with PMAxx-ddPCR in dairy products

One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log10 copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food.

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media.Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling.Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture.These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.

Simultaneous Detection of Three Foodborne Pathogens Based on Immunomagnetic Nanoparticles and Fluorescent Quantum Dots.

This paper presents a peptide-mediated immunomagnetic separation technique and an immunofluorescence quantum dot technique for simultaneous and rapid detection of Escherichia coliO157:H7, Staphylococcus aureus, and Vibrio parahaemolyticus. First, three peptides that can specifically recognize the three foodborne pathogens were combined with magnetic nanoparticles to form an immunomagnetic nanoparticle probe for capturing three kinds of target bacteria and then added three quantum dot probes (quantum dots-aptamer), which formed a sandwich composite structure. When the three quantum dot probes specifically combined with the three pathogenic bacteria, the remaining fluorescent signal in the supernatant will be reduced by magnetic separation. Therefore, the remaining fluorescent signal in the supernatant can be measured with a fluorescence spectrophotometer to indirectly determine the three pathogens in the sample. The linear range of the method was 10–107 cfu/mL, and in the buffer, the detection limits of E. coliO157:H7, S. aureus, and V. parahaemolyticus were 2.460, 5.407, and 3.770 cfu/mL, respectively. In the tap water simulation, the detection limits of E. coliO157:H7, S. aureus, and V. parahaemolyticus were 2.730, 1.990 × 101, and 4.480 cfu/mL, respectively. In the milk simulation sample, the detection limits of E. coliO157:H7, S. aureus, and V. parahaemolyticus were 6.660, 1.070 × 101, and 2.236 × 101 cfu/mL, respectively. The method we presented can detect three kinds of foodborne pathogens at the same time, and the entire experimental process did not exceed 4 h. It has high sensitivity and low detection limit and may be used in the sample detection of other pathogens.

Antibody-modified magnetic nanoparticles as specific high-efficient cell-separation agents.

Separation of tumor cells is a promising approach that helps not only in early detection of cancer but also as an efficient tool that holds great importance in prohibiting cancer cell mutation, drug resistance to treatments, and in granting successful adjuvant therapies. As one of the highly efficient processes for the separation of single cells, tumor cells, and specific proteins from fresh whole blood, a magnetic iron oxide nanoparticle (IONP)-based immunomagnetic separation technique has been developed in this article. The synthesized IONPs were modified with antibodies (Abs) against human epithelial growth factor receptor 2 (HER2), which is overexpressed and/or amplified in about 15% of breast cancer patients with several types of human cancer cells. The prepared Ab-conjugated IONPs (Ab-IONPs) attach HER2-positive cancer cells exclusively and can serve as specific high-efficient single-cell separation agents. The results showed that the magnetic IONPs have been successfully attached to the Abs via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide linkers. Maximum targeting efficiency of the Ab-IONP complex, which was 94.5 ± 0.8% for BT474 and 70.6 ± 0.4% for mixture of cells (BT474 and MCF7), was achieved with a minimum amount of Abs, to provide an economically efficient single-cell detection device.

Sperm fishing: antigen-antibody complexes for the capture and enrichment of spermatozoa in mixed cell substrates

ABSTRACTIn many sexual assault cases, the most common evidence gathered comes in the form of intimate swabs. These swabs contain cells from both the female and male. In these cases the most common cell present is epithelial cells from the female with relatively few spermatozoa from the male. To produce an STR DNA profile suitable for an investigation, separation of the cell types is required to prevent a mixed profile that may complicate interpretation. While there are methods that can achieve this separation, they are labour intensive and have no guarantee of complete separation of the spermatozoa. Immunomagnetic bead separation techniques are being adapted from other scientific fields to be used in forensic biology to separate spermatozoa from mixed substrates. This study tested the binding of a Hyaluronidase PH-20 (SPAM1) antibody to spermatozoa in mixed cell samples that have been dried and stored for a short period of time. Fluorescently tagged antibodies were incubated with cellular mixtures and visualized using fluorescent microscopy. Successful binding of the antibodies to spermatozoa was observed in all samples. The SPAM1 antibody appears to be suitable for selection of spermatozoa and creates opportunities for linking with magnetic beads facilitate cell separation and DNA profiling.

Akciğer kanseri hastalarında dolaşımdaki tümör hücrelerini nasıl tespit ederiz? akım sitometrisi ile yapılan kısa bir çalışma

Circulating tumor cells (CTCs) play a crucial role in the metastatic spread of carcinoma. Therefore, CTC has been interest of a subject in the past few decades in terms of prognosis and response to the therapy in several cancer diseases. Recent improvements in technical approaches maintain to identify CTCs from whole blood have demonstrated the potential value of CTC detection as a liquid biopsy especially in those tumors where tissue accessibility is often challenging as in lung cancer. Lung cancer is the most common cause of death from cancer worldwide in both men and women which is commonly metastasize before it is diagnosed. The aim of this study is to enumerate of CTCs in peripheral blood sample (7.5 mL) of lung cancer patients by flow cytometry. Our modified method which consists of enrichment and detection steps get involved in 9 patients with lung cancer and 9 healthy volunteers. We performed a density-based ficoll gradient centrifugation and a immunomagnetic separation technique (CD45 negative selection) for the enrichment step. Next, multi-parameter flow cytometry based on the expression of anti-epithelial cell adhesion molecule and cytokeratins was used to detect circulating tumor cells among enriched cells. According to our results, circulating tumor cells were not detected on healthy volunteers but circulating tumor cells were found in all of patients with lung cancer (Z=3.823; p<0.001). We demonstrate that circulating tumor cells were detectable in peripheral blood sample of lung cancer patients by our modified method.

Community-based surveillance of Cryptosporidium in the indigenous community of Boliwong, Philippines: from April to December 2017.

OBJECTIVESFor the first time, Boliwong, an indigenous community in the Philippines, was surveyed for the prevalence of Cryptosporidium from April to December 2017.METHODSCryptosporidium oocysts were detected in samples from the river, creek, and water pumps via immunomagnetic separation techniques, and from human and animal concentrated faecal samples using the modified Ziehl-Neelsen technique.RESULTSSeven of the 24 water samples (29.2%) were positive for Cryptosporidium, with the highest concentration (0.8 oocyst/L) detected in the creek. Of 35 fecal samples from different animal groups, 8 (21.6%) were positive for Cryptosporidium oocysts. The highest intensity of oocyst shedding was detected in dogs (χ2 =8.00). Of the 137 human fecal samples, 39 (28.5%) were infected with Cryptosporidium. In this study, 3 risk factors were found to be associated with infection: (1) location (crude odds ratio [cOR], 16.39; 95% confidence interval [CI], 2.11 to 127.41; p=0.008), (2) drinking water from the natural spring (cOR, 0.29; 95% CI, 0.11 to 0.82; p<0.05), and (3) using an open pit as a sanitary toilet facility (cOR, 2.44; 95% CI, 1.14 to 5.20; p<0.05). When the cOR was adjusted, using an open pit as a sanitary toilet facility remained a significant risk factor of infection (adjusted OR, 0.41; 95% CI, 0.19 to 0.90; p<0.05).CONCLUSIONSThere is a potentially emerging Cryptosporidium zoonosis in Boliwong, Lagawe, Philippines. It is recommended that the toilet facilities and the water system in the community be rehabilitated to avoid any possible disease outbreak. Health education is also needed in the community to maintain proper hygiene and sanitation practices.

Circulating endothelial cells and microparticles as diagnostic and prognostic biomarkers in small-cell lung cancer

ObjectivesIt has been proposed that circulating endothelial cells (CECs) and microparticles (MPs) may be useful for the assessment of patients with non-small-cell lung cancer (NSCLC). However, little is known about the potential clinical relevance of these biomarkers in small-cell lung cancer (SCLC). Therefore, we investigated the utility of baseline levels of CECs and MPs in SCLC patients. Materials and methodsAn immunomagnetic separation (IMS) technique was used to isolate and quantify CECs in the peripheral blood, while plasma samples were analyzed using flow cytometry for the measurement of circulating MPs. ResultsWe prospectively collected data from 56 patients and 41 healthy individuals. Forty-three patients presented at initial diagnosis and 13 patients presented at relapse. Baseline levels of CECs and MPs were significantly higher in SCLC patients either at initial diagnosis or at relapse than in healthy subjects (p < 0.0002 and p < 0.007, respectively). However, estimated tumor volume (ETV) was significantly correlated with basal MP values (p < 0.0001) but not with pretreatment CECs (p = 0.57). The amount of baseline CECs and MPs was significantly lower in patients with an objective response (OR, n = 23) than in those with progressive disease (PD, n = 15) after treatment (p = 0.016 and 0.05, respectively). With cut-off values of 110 cells/mL for CECs and 1257 events/μL for MPs according to receiver operating characteristics (ROC) analysis, baseline levels of these biomarkers were not significantly correlated with either progression-free survival (PFS) or overall survival (OS). However, patients with 6-month PFS displayed significantly decreased pretreatment CEC counts (p = 0.042), whereas basal MP values significantly increased in 1-year survivors compared with those in non-survivors (p = 0.05). ConclusionOur results suggest that baseline CECs and MPs may be predictive biomarkers of tumor response and long-term survival in SCLC patients.

Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains

BackgroundLegionella pneumophila, the causative agent of Legionnaire’s disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water.ResultsWith ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb- stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population.ConclusionsThe results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.

Therapeutic Effect of CD4+CD25+ Regulatory T Cells Amplified In Vitro on Experimental Autoimmune Neuritis in Rats

Background/Aims: This study aimed to explore whether the adoptive transfusion of autologous CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) has a therapeutic effect on Experimental autoimmune neuritis (EAN) model rats, and it provides new experimental and theoretical bases for the immunotherapy of Guillain-Barre syndrome (GBS). Methods: CD4+CD25+ Tregs were sorted from the spleens of rats using immunomagnetic bead separation techniques combined with flow cytometry. Their in vitro inhibitory function was determined using a lymphocyte proliferation inhibition test, and their purity was confirmed by flow cytometry. Cells were stimulated using CD3/CD28 monoclonal antibodies and were cultured in culture medium containing interleukin 2 (IL-2), transforming growth factor-β (TGF-β) and rapamycin. After 15 days of amplification, CD4+CD25+ Tregs were collected and transfused into EAN model rats. Changes in the pathology and electron microscopical morphology of rat sciatic nerves in the normal group, untreated group, low-dose group (2 × 10⁷) and high-dose group (4 × 10⁷) were observed, and the expression of CD4+CD25+FOXP3 in peripheral blood in the four groups of rats was detected by flow cytometry. Results: Compared with rats in the untreated group, rats in the treatment groups had significantly reduced infiltration of inflammatory cells in the sciatic nerve, as well as myelin and axonal damage. Additionally, the CD4+CD25+ Tregs levels in peripheral blood were significantly higher than those in the untreated group (P< 0. 05). Moreover, the therapeutic effect became more significant with an increase in the dose of adoptive transfusion. Conclusion: Adoptive transfusion of CD4+CD25+ Tregs into EAN model rats has significant therapeutic effects.

Circulating endothelial cells and microparticles for prediction of tumor progression and outcomes in advanced non-small cell lung cancer.

Circulating endothelial cells (CECs) and microparticles (MPs) are proposed as useful biosensors for angiogenesis and membrane damage in cancer. We investigated their predictive value for progression disease (PD) and clinical outcomes in advanced non-small cell lung cancer (NSCLC) patients treated with cytotoxic chemotherapy. Peripheral blood samples were obtained from 60 patients. Immunomagnetic separation (IMS) and flow cytometry techniques were used to quantify CECs and MPs, respectively. Receiver operating characteristics (ROC) analysis was used to determine the optimal cutoff values for CECs and MPs counts according to their levels in patients with an objective response (OR) and non-responders after treatment. Baseline serum biomarkers levels and their kinetics after chemotherapy were correlated with tumor response and outcomes in advanced NSCLC patients. Forty-seven patients presented an OR after chemotherapy. Of these, 28 patients progressed within three months. Through an increase in their levels during or after chemotherapy, CECs and MPs correctly predicted PD in 57% and 61% of these patients, respectively. Regarding tumor stage, NSCLC patients with stage IV had significantly higher pretreatment CECs and MPs levels than stage III patients (p= 0.037 and 0.018, respectively). Moreover, progression-free survival (PFS) was significantly longer in patients with high baseline CECs levels than those with low pretreatment CECs values (p= 0.05). Moreover, patients with high percentage change in CECs count after chemotherapy had significantly longer time to progression (TTP) duration (p= 0.018). Our findings suggest the increase in CECs and MPs number during or after chemotherapy as predictive biomarkers of tumor progression in advanced NSCLC patients. An association of basal CECs and MPs values with tumor stage was also shown in advanced NSCLC patients. However, baseline CECs levels and their kinetics after chemotherapy seem to be prognostic factors in advanced NSCLC.

First molecular characterization of Cryptosporidium from three different points of two main rivers in Kuantan, Malaysia using 18S rRNA gene nested PCR

Objective: To identify 18S ribosomal RNA partial sequences from three different points, namely downstream, midstream and upstream of two water rivers in Kuantan, Pahang, Malaysia Methods: In this study, a total of six water samples were collected from three different points of both Kuantan River and Balok River. Each water sample was processed and concentrated using continuous flow centrifuge (CFC) machine and purified using immunomagnetic separation (IMS) technique. Nested Polymerase Chain Reaction (nPCR) was applied to detect 18S ribosomal RNA sequence in those samples after DNA extraction. Genotyping and phylogenetic analysis were performed on the basis of 18S rDNA gene sequence of Cryptosporidium. Results: A total of five samples from six different points of both Kuantan River and Balok River were contaminated with Cryptosporidium. Only river water sample from the upstream point of Kuantan River was negative for Cryptosporidium. In this study, the sequencing results of five samples from both Kuantan River and Balok River belonged to Cryptosporidium parvum (C. parvum). Conclusions: To the best of our knowledge, this is the first report of C. parvum genotyping in both Kuantan River and Balok River by using molecular approach nested polymerase chain reaction (nPCR) on 18S ribosomal RNA gene.

The association of Cryptosporidium from three different points of Balok River and Kuantan River by using physico-chemical and heavy metal assessments

Objective: The objectives of this study were to detect the occurrence of Cryptosporidium oocysts and to assess the physico-chemical and heavy metal parameters in two main rivers in Kuantan. Methods: Water samples were collected at three sampling points per river (upstream, midstream, downstream) from Kuantan River and Balok River. Samples were filtered using the Continuous Flow Centrifugation (CFC) machine followed by immunomagnetic separation technique to isolate Cryptosporidium oocysts and stained using fluorescein isothiocyanate (FITC) –staining. Cryptosporidium oocysts were examined using fluorescence microscope. Physical parameters were assessed in-situ using Cyber Scan PCD 650 multi-parameter instrument. Both chemical and heavy metal assessment were done in the laboratory following the American Public Health Association (APHA) Standard Method with slight modification. The parameters attained were compared with the Interim National Water Quality Standards (INWQS) which is the standard vital parameters used to evaluate the safety level of surface water in Malaysia. Results: All samples were positive with Cryptosporidium oocysts. Results for physical parameters were within the range of Interim National Water Quality Standards (INWQS) in Malaysia. For chemical assessment, results for chemical oxygen demand and biological oxygen demand exceeded the INWQS range in most of the sampling points. Of 23 metal elements assessed, only 9 elements were found. Both Kuantan River and Balok River can be classified under Class III river following INWQS which requires extensive treatment. Conclusions: This study hoped to provide new and updated information on the occurrence of Cryptosporidium and its physico-chemical assessment in two main rivers in Kuantan. Future study on molecular identification of Cryptosporidium in rivers needs to be done in order to identify the source of transmission of this waterborne parasite.

Rapid detection of Listeria monocytogenes using fluorescence immunochromatographic assay combined with immunomagnetic separation technique

SummaryTo ensure the safety and quality of food ingredients, especially meat and dairy products, a high‐throughput, rapid and sensitive method to detect Listeria monocytogenes (LM) is always on a high demand. In this work, a specific induction method to enrich and detect LM based on fluorescence immunochromatographic assay (FICA) combined with immunomagnetic separation (IMS) techniques has been developed. The immunomagnetic‐beads (IM) beads were obtained through functionalised magnetic microspheres and the conjugated reaction between the carboxyl on beads surface and amino groups of antibody. The prepared IM‐beads could be used for rapidly enriching pathogenic bacteria with fewer steps, resulting in a high specificity for four pathogenic serotypes and about a 40‐fold improvement of detection limit compared with FICA only. In addition, this method was successfully applied in LM detection in sausage, pork and milk samples with a potential for further application in rapid on‐site detection of pathogenic bacteria.

Lectin-magnetic separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR

Although standard methods for analyzing water samples for the protozoan parasites Cryptosporidium spp. and Giardia duodenalis are available and widely used, equivalent methods for analyzing water samples for Toxoplasma gondii oocysts are lacking. This is partly due to the lack of a readily available, reliable immunomagnetic separation technique (IMS). Here we investigated the use of lectin-magnetic separation (LMS) for isolating T. gondii oocysts from water sample concentrates, with subsequent detection by microscopy or molecular methods. Four different types of magnetic beads coated with wheat germ agglutinin (WGA) were tested for capture of oocysts from clean or dirty water samples. Dynabeads (Myone T1 and M-280) consistently provided mean capture efficiencies from 1 ml clean water in excess of 97%. High recoveries were also found with Tamavidin beads (in excess of 90%) when LMS was used for capture from a small (1 ml) volume. Dissociation (required for detection by microscopy) using 0.1N hydrochloric acid (HCl), as standard in IMS, was not successful, but could be achieved using a combination of acidified pepsin (AP) and N-acetyl d-glucosamine. Although simple centrifugation was as effective as LMS when concentrating high numbers of oocysts from clean water, LMS provided superior results when oocysts numbers were low or the water sample was dirty. Application of LMS integrated with qPCR enabled detection of 10 oocysts per 10 ml dirty water sample concentrate. These findings indicate that LMS with WGA coupled to magnetic beads could be an efficient isolation step in the analysis of water sample concentrates for T. gondii oocysts, with detection either by microscopy or by qPCR.