Search Results (13)

Legionella pneumophila are pathogenic bacteria that repeatedly occur in high concentrations in the process water of evaporative cooling systems (ECS). When released into the environment, the resulting bioaerosols can cause outbreaks with fatal consequences. The official, internationally accepted detection method for Legionella spp. in water samples is based on cultivation. However, cultivation is time-consuming and may underestimate the total count of viable L. pneumophila in ECS. Therefore, culture-independent methods are receiving attention for rapid monitoring. Cartridge-based immunomagnetic separation (IMS) coupled with flow cytometry (FCM) is an innovative, antibody-based method for the culture-independent quantification of L. pneumophila, using a panel of antibodies against serogroup (Sg) 1–15. We characterized the IMS-FCM method as a quantitative rapid test by general analytical procedures. Viable cryopreserved L. pneumophila standards were used in calibration experiments for the method. We achieved detection limits for Sg 1, Sg 4, and Sg 6 of 100, 105 and 88 viable cells per 100 mL, respectively. Furthermore, we demonstrated the practical applicability of IMS-FCM with real ECS samples and compared the performance against cultivation. Cultivation showed here no positive results, but IMS-FCM evidenced L. pneumophila in a range of 0–80,000 viable cells per 100 mL. This work demonstrates that IMS-FCM is a suitable, culture-independent, quantitative method for rapidly monitoring L. pneumophila. Full article

Pathogen detection is still a challenging issue for public health, especially in food products. A selective preconcentration step is also necessary if the target pathogen concentration is very low or if the sample volume is limited in the analysis. Plate counting (24–48 h) methods should be replaced by novel biosensor systems as an alternative reliable pathogen detection technique. The usage of a capillary-driven microfluidic chip is an alternative method for pathogen detection, with the combination of surface-enhanced Raman scattering (SERS) measurements. Here, we constructed microchambers with capillary microchannels to provide nanoparticle–pathogen transportation from one chamber to the other. Escherichia coli (E. coli) was selected as a model pathogen and specific antibody-modified magnetic nanoparticles (MNPs) as a capture probe in a complex milk matrix. MNPs that captured E. coli were transferred in a capillary-driven microfluidic chip consisting of four chambers, and 4-aminothiophenol (4-ATP)-labelled gold nanorods (Au NRs) were used as the Raman probe in the capillary-driven microfluidic chip. The MNPs provided immunomagnetic (IMS) separation and preconcentration of analytes from the sample matrix and then, 4-ATP-labelled Au NRs provided an SERS response by forming sandwich immunoassay structures in the last chamber of the capillary-driven microfluidic chip. The developed SERS-based method could detect 10¹–10⁷ cfu/mL of E. coli with the total analysis time of less than 60 min. Selectivity of the developed method was also tested by using Salmonella enteritidis (S. enteritidis) and Staphylococcus aureus (S. aureus) as analytes, and very weak signals were observed. Full article

Rapid and accurate food pathogen detection is an essential step to preventing foodborne illnesses. Before detection, removal of bacteria from the food matrix and concentration to detectable levels are often essential steps. Although many reviews discuss rapid concentration methods for foodborne pathogens, the use of glycan-coated magnetic nanoparticles (MNPs) is often omitted. This review seeks to analyze the potential of this technique as a rapid and cost-effective solution for concentration of bacteria directly from foods. The primary focus is the mechanism of glycan-coated MNP binding, as well as its current applications in concentration of foodborne pathogens. First, a background on the synthesis, properties, and applications of MNPs is provided. Second, synthesis of glycan-coated particles and their theorized mechanism for bacterial adhesion is described. Existing research into extraction of bacteria directly from food matrices is also analyzed. Finally, glycan-coated MNPs are compared to the magnetic separation technique of immunomagnetic separation (IMS) in terms of cost, time, and other factors. At its current state, glycan-coated MNPs require more research to fully identify the mechanism, potential for optimization, and extraction capabilities directly in food matrices. However, current research indicates glycan-coated MNPs are an incredibly cost-effective method for rapid food pathogen extraction and concentration. Full article

This work addresses a method that combines immunomagnetic separation (IMS) and paper-based nucleic acid immunochromatographic assay for the sensitive detection of Mycolicibacterium fortuitum (basonym Mycobacterium fortuitum) In particular, the preconcentration of the bacteria was achieved by using magnetic particles modified with an antibody specific towards mycobacteria. Following the IMS, the bacteria were lysed, and the genome was amplified by double-tagging PCR, using a set of primers specific for the 16S rRNA gene for Mycobacterium. During the amplification, the amplicons were labeled with biotin and digoxigenin tags. Moreover, a comparative study of paper-based immunochromatographic platforms, relying on vertical and lateral flow and on the use of streptavidin gold nanoparticles as a signal generating system, was also performed. The visual readout was achieved when the gold-modified amplicons were captured by the anti-DIG antibody in the test line. The analytical performance of both methods, nucleic acid vertical flow (NAVF) and nucleic acid lateral flow (NALF), is also discussed. Although NALF showed lower limit of detections (LODs), both NALF and NAVF combined with IMS were able to detect the required LOD in hemodialysis water, becoming two promising and useful techniques for the rapid screening of water supplies in hemodialysis centers, to prevent the exposure of immunosuppressed patients to contaminated sources. Full article

The main goal of this study was to apply magnetic bead surface functionalization in the form of immunomagnetic separation (IMS) combined with real-time polymerase chain reaction (qPCR) (IMS-qPCR) to detect Human mastadenovirus species C (HAdV-C) and F (HAdV-F) in water samples. The technique efficiency was compared to a nonfunctionalized method (ultracentrifugation) followed by laboratory detection. Tests were carried out to standardize IMS parameters followed by tests on 15 water samples concentrated by IMS and ultracentrifugation. Microscopic analyses detected a successful beads–antibody attachment. HAdV was detected up to dilutions of 10⁻⁶ by IMS-qPCR, and samples concentrated by IMS were able to infect cell cultures. In water samples, HAdV-C was detected in 60% (monoclonal) and 47% (polyclonal) by IMS-qPCR, while 13% of samples concentrated by ultracentrifugation gave a positive result. HAdV-F was positive in 27% of samples by IMS-qPCR (polyclonal) and ultracentrifugation and 20% by IMS-qPCR (monoclonal). The rate of detection varied from 4.55 × 10² to 5.83 × 10⁶ genomic copies/L for IMS-qPCR and from 2.00 × 10² to 2.11 × 10³ GC/L for ultracentrifugation. IMS showed to be a more effective concentration technique for HAdV than ultracentrifugation, improving the assessment of infectious HAdV in water resources. Full article

In this study, we describe the development of an automated immunomagnetic separation device combined with real-time polymerase chain reaction (PCR) for detecting foodborne bacteria. Immunomagnetic separation (IMS) is a well-known method for the separation and concentration of target bacteria from a large volume of food samples. Magnetic beads functionalized with an antibody provide selectivity for target bacteria such as Escherichia coli O157:H7. Moreover, compared to conventional methods, real-time PCR enables high-sensitivity detection of target bacteria. The method proposed in this study involves three steps: (1) pre-enrichment, (2) automated IMS and concentration of target bacteria, and (3) detection of target bacteria by real-time PCR. Using food samples with a working sample volume as large as 250 mL, the whole process only requires 3 h. As a result, target bacteria in the range of 10¹–10² colony-forming units per mg or g of sample can be detected in food samples, such as milk, ground beef, and cabbage, by using the proposed approach. We anticipate that the automated IMS system combined with real-time PCR will contribute to the development of a fully automated system for detecting foodborne bacteria and serve as a multi-tester for a variety of bacterial strains in the capacity of a sample-to-answer device in the near future. Full article

A new generation of magnetic lateral flow immunoassays is emerging as powerful tool for diagnostics. They rely on the use of magnetic nanoparticles (MNP) as detecting label, replacing conventional gold or latex beads. MNPs can be sensed and quantified by means of external devices, allowing the development of immunochromatographic tests with a quantitative capability. Moreover, they have an added advantage because they can be used for immunomagnetic separation (IMS), with improvements in selectivity and sensitivity. In this paper, we have reviewed the current knowledge on magnetic-lateral flow immunoassay (LFIA), coupled with both research and commercially available instruments. The work in the literature has been classified in two categories: optical and magnetic sensing. We have analysed the type of magnetic nanoparticles used in each case, their size, coating, crystal structure and the functional groups for their conjugation with biomolecules. We have also taken into account the analytical characteristics and the type of transduction. Magnetic LFIA have been used for the determination of biomarkers, pathogens, toxins, allergens and drugs. Nanocomposites have been developed as alternative to MNP with the purpose of sensitivity enhancement. Moreover, IMS in combination with other detection principles could also improve sensitivity and limit of detection. The critical analysis in this review could have an impact for the future development of magnetic LFIA in fields requiring both rapid separation and quantification. Full article

The aim of this study was to evaluate the occurrence of Cryptosporidium oocysts in a drinking water treatment plant (DWTP) located in a rural area of northern Italy. Influent and effluent samples were collected at the DWTP over three years (2013–2016). In parallel, tap water samples from a public drinking fountain were collected as well. All samples were analyzed for the presence of Cryptosporidium spp. oocysts by a common method based on an immunomagnetic separation (IMS)/immunofluorescence assay (IFA), complemented by 4,6-diamidino-2-phenylindole (DAPI) staining. A reverse transcriptase-PCR (RT-PCR) protocol was added to evaluate oocyst viability. The results highlighted a high variability of oocyst concentrations across all samples (mean 4.3 ± 5.8/100 L) and a high variability in the percentage of DAPI-positive specimens (mean 48.2% ± 40.3%). Conversely, RT-PCR did not reveal the presence of viable C. parvum and C. hominis oocysts. A nested PCR targeting Cryptosporidium 18S ribosomal DNA, carried out in two water samples, confirmed the presence of a Cryptosporidium genotype associated with wild animals in the river and in tap water. The results obtained underline the vulnerability of the investigated surface water to Cryptosporidium spp. contamination. Although the recovered Cryptosporidium genotype is not a human pathogen, its presence demonstrates the existence of a potential pathogen Cryptosporidium spp. contamination risk. Moreover, these results underline the importance of also considering unconventional (not bacterial) biological contaminations (protozoa) in water resources in rural areas, including those of developed countries. Full article

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample. Full article

The occurrence of waterborne parasites coupled with water parameters at various processing sites of two drinking water treatment plants (A and B) and seven distribution system (DS) sites in Sarawak, Malaysia were studied. Ten liters of water underwent immunomagnetic separation (IMS) technique to detect the presence of Giardia and Cryptosporidium (oo)cysts. The remaining supernatant was used to detect other parasites whilst 50 mL of water sample was each used in the detection of free-living amoebae and fecal coliforms. Sampled water was positive for Giardia (32.9%; 28/85), Cryptosporidium (18.8%; 16/85) followed by Spirometra ova-like (25.9%; 22/85), Blastocystis-like (25.9%; 22/85), nematode larvae-like (8.2%; 7/85) and Taenia ova-like (1.2%; 1/85). Meanwhile, 90.2% (55/61) samples were positive for Acanthamoeba and Naegleria via cultivation and of these, 11 isolates were confirmed as Acanthamoeba genotype T3 (5/7) and T4 (2/7) followed by Naegleria sp. (4/11), Naegleria italica (2/11), Naegleria australiensis (1/11), Naegleria angularis (1/11) and Vahlkampfia sp. (3/11). Cryptosporidium, Acanthamoeba and Naegleria were also detected in one of the seven tested DS sites. Only Giardia and Cryptosporidium showed significant correlations with fluoride and fecal coliforms. These results describe the occurrence of waterborne parasites that will assist key stakeholders in mitigating contamination at the specific sites. Full article

In this study, hollow-fiber ultrafiltration (UF) was assessed for recovery of Escherichia coli, Clostridium perfringens spores, Cryptosporidium parvum oocysts, echovirus 1, and bacteriophages MS2 and ΦX174 from ground and surface waters. Microbes were seeded into twenty-two 50-L water samples that were collected from the Southeastern United States and concentrated to ~500 mL by UF. Secondary concentration was performed for C. parvum by centrifugation followed by immunomagnetic separation. Secondary concentration for viruses was performed using centrifugal ultrafilters or polyethylene glycol precipitation. Nine water quality parameters were measured in each water sample to determine whether water quality data correlated with UF and secondary concentration recovery efficiencies. Average UF recovery efficiencies were 66%–95% for the six enteric microbes. Average recovery efficiencies for the secondary concentration methods were 35%–95% for C. parvum and the viruses. Overall, measured water quality parameters were not significantly associated with UF recovery efficiencies. However, recovery of ΦX174 was negatively correlated with turbidity. The recovery data demonstrate that UF can be an effective method for concentrating diverse microbes from ground and surface waters. This study highlights the utility of tangential-flow hollow fiber ultrafiltration for recovery of bacteria, viruses, and parasites from large volume environmental water samples. Full article

Food defense requires the means to efficiently screen large volumes of food for microbial pathogens. Even rapid detection methods often require lengthy enrichment steps, making them impractical for this application. There is a great need for rapid, sensitive, specific, and inexpensive methods for extracting and concentrating microbial pathogens from food. In this study, an immuno-magnetic separation (IMS) methodology was developed for Escherichia coli O157:H7, using electrically active magnetic nanoparticles (EAMNPs). The analytical specificity of the IMS method was evaluated against Escherichia coli O55:H7 and Shigella boydii, and was improved over previous protocols by the addition of sodium chloride during the conjugation of antibodies onto MNPs. The analytical sensitivity of the IMS method was greatest when a high concentration of antibodies (1.0 mg/mL) was present during conjugation. EAMNP concentrations of 1.0 and 0.5 mg/mL provided optimal analytical sensitivity and analytical specificity. The entire IMS procedure requires only 35 min, and antibody-conjugated MNPs show no decline in performance up to 149 days after conjugation. This analytically sensitive and specific extraction protocol has excellent longevity and shows promise as an effective extraction for multiple electrochemical biosensor applications. Full article

Salmonella spp. is associated with fecal pollution and capable of surviving for long periods in aquatic environments. Instead of the traditional, time-consuming biochemical detection, polymerase chain reaction (PCR) allows rapid identification of Salmonella directly concentrated from water samples. However, prevalence of Salmonella may be underestimated because of the vulnerability of PCR to various environmental chemicals like humic acid, compounded by the fact that various DNA polymerases have different susceptibility to humic acid. Because immunomagnetic separation (IMS) theoretically could isolate Salmonella from other microbes and facilitate removal of aquatic PCR inhibitors of different sizes, this study aims to compare the efficiency of conventional PCR combined with immunomagnetic separation (IMS) for Salmonella detection within a moderately polluted watershed. In our study, the positive rate was increased from 17.6% to 47% with nearly ten-fold improvement in the detection limit. These results suggest the sensitivity of Salmonella detection could be enhanced by IMS, particularly in low quality surface waters. Due to its effects on clearance of aquatic pollutants, IMS may be suitable for most DNA polymerases for Salmonella detection. Full article