Detection Of Waterborne Pathogens Research Articles

Fast, sensitive, and reliable detection of waterborne pathogens by digital PCR after coagulation and foam concentration

The quantification of pathogens is important for assessing water safety and preventing disease outbreaks. Culture-independent approaches, such as quantitative PCR (qPCR) and digital PCR (dPCR), are useful techniques for quantifying pathogens in water samples. However, since pathogens are usually present at low concentrations in water, it is necessary to concentrate microbial cells before extracting their DNA. Many existing microbial concentration methods are inefficient or take a long time to perform. In this study, we applied a coagulation and foam separation method to concentrate environmental water samples of between 1000 and 5000mL to 100μL of DNA (i.e., a 1-5×104-fold concentration). The concentration process took <1h. The DNA samples were then used to quantify various target pathogens using dPCR. One gene, the Shiga toxin gene (stx2) of Shiga toxin-producing Escherichia coli, was detected at 32 copies/100mL in a river water sample. The coagulation and foam concentration method followed by dPCR reported herein is a fast, sensitive, and reliable method to quantify pathogen genes in environmental water samples.

Synthesis and application of superabsorbent polymer microspheres for rapid concentration and quantification of microbial pathogens in ambient water

Even though numerous methods have been developed for the detection and quantification of waterborne pathogens, the application of these methods is often hindered by the very low pathogen concentrations in natural waters. Therefore, rapid and efficient sample concentration methods are urgently needed. Here we present a novel method to pre-concentrate microbial pathogens in water using a portable 3D-printed system with super-absorbent polymer (SAP) microspheres, which can effectively reduce the actual volume of water in a collected sample. The SAP microspheres absorb water while excluding bacteria and viruses by size exclusion and charge repulsion. To improve the water absorption capacity of SAP in varying ionic strength waters (0–100 mM), we optimized the formulation of SAP to 180 g⋅L−1 Acrylamide, 75 g⋅L−1 Itaconic Acid and 4.0 g⋅L−1 Bis-Acrylamide for the highest ionic strength water as a function of the extent of cross-linking and the concentration of counter ions. Fluorescence microscopy and double-layer agar plating respectively showed that the 3D-printed system with optimally-designed SAP microspheres could rapidly achieve a 10-fold increase in the concentration of Escherichia coli (E. coli) and bacteriophage MS2 within 20 min with concentration efficiencies of 87% and 96%, respectively. Fold changes between concentrated and original samples from qPCR and RT-qPCR results were found to be respectively 11.34–22.27 for E. coli with original concentrations from 104 to 106 cell·mL−1, and 8.20–13.81 for MS2 with original concentrations from 104 to 106 PFU·mL−1. Furthermore, SAP microspheres can be reused for 20 times without performance loss, significantly decreasing the cost of our concentration system.

Recreational water monitoring: Nanofluidic qRT‐PCR chip for assessing beach water safety

AbstractImproved recreational water monitoring using rapid molecular genetic methods would decrease both public health risks and unnecessary beach closures. Hence, we developed a novel nanofluidic quantitative real‐time PCR (qPCR) in the OpenArray platform to (a) detect and quantify fecal indicator bacteria (FIBs; N = 2), (b) identify contaminant sources (microbial source tracking (MST); N = 7), and (c) detect human virulent pathogens (virulence gene markers; N = 15). Our water monitoring OpenArray plate reliably detects as few as two template copies/hole (OpenArray® well), with some marker sensitivities as low as single‐copy detection. The OpenArray plate showed high target sequence specificity and returned expected patterns of contaminants for fecal and sewage samples. We found Canada geese and seagulls were the leading causes of contamination at beaches that tested positive for coliforms. When we incorporated robotic DNA extraction, we were able to process samples from water to FIBs, MST, and waterborne pathogen detection and quantification within four hours. Our monitoring TaqMan qPCR assays in the OpenArray platform is uniquely valuable for regulatory agencies charged with beach water safety as well as for researchers interested in human health implications of aquatic microbial community structure.

Life cycle analysis applied to analytical methods for the detection of waterborne pathogens

The Carbon Footprint (CFP) of novel analytical methods for waterborne pathogens detection is calculated to evaluate their environmental impact compared to the conventional methods used so far in drinking water analysis. These new-flanged methods are developed under the EU project Aquavalens that has as objective the development of sustainable technologies to provide water system managers with tools to better control the safety of water supplies and their impact on public health.The sources of primary data for the CFP calculation were the project partners, who were at same time part of the non-expert audience of the Life Cycle Assessment to whom the results are addressed. The new analytical methods are based on the use of two types of pathogen detection products, hereinafter referred to as analytical products: an on-line monitoring device and two ready-to-use kits for molecular detection techniques. These products were designed and manufactured by the producer partners of the project and they were tested experimentally in six European locations by different end-users partners. Specific questionnaires were designed to obtain flows of materials, energy and wastes associated to the analysis of one water sample, with special attention to packaging in the production of the analytical products and transport and energy consumption during the analytical procedure conducted.The use phase was identified as the major contributor to the CFP along the whole life cycle of the new analytical products. Up to 66% reduction was calculated for the Aquavalens analytical methods in relation to conventional methods when pathogens from the three groups (bacteria, protozoa and viruses) were analysed. The parametric variability calculated through Monte Carlo simulation showed the effect of different praxis and management during sampling and laboratory work. The collection of the samples, and specifically the transport to and from the sampling points to the laboratory, was found to be the most determinant contributor in the total CFP. Besides, the analysis considered alternative scenarios for the type of vehicle used in the sampling transport and different electricity production mixes.

Fluorescent beads-based biosensors for multiplexed detection of waterborne pathogens

Fluorescent beads-based biosensors for multiplexed detection of waterborne pathogens

Electrochemical Methodologies for the Detection of Pathogens

Bacterial infections remain one of the principal causes of morbidity and mortality worldwide. The number of deaths due to infections is declining every year by only 1% with a forecast of 13 million deaths in 2050. Among the 1400 recognized human pathogens, the majority of infectious diseases is caused by just a few, about 20 pathogens only. While the development of vaccinations and novel antibacterial drugs and treatments are at the forefront of research, and strongly financially supported by policy makers, another manner to limit and control infectious outbreaks is targeting the development and implementation of early warning systems, which indicate qualitatively and quantitatively the presence of a pathogen. As toxin contaminated food and drink are a potential threat to human health and consequently have a significant socioeconomic impact worldwide, the detection of pathogenic bacteria remains not only a big scientific challenge but also a practical problem of enormous significance. Numerous analytical methods, including conventional culturing and staining techniques as well as molecular methods based on polymerase chain reaction amplification and immunological assays, have emerged over the years and are used to identify and quantify pathogenic agents. While being highly sensitive in most cases, these approaches are highly time, labor, and cost consuming, requiring trained personnel to perform the frequently complex assays. A great challenge in this field is therefore to develop rapid, sensitive, specific, and if possible miniaturized devices to validate the presence of pathogens in cost and time efficient manners. Electrochemical sensors are well accepted powerful tools for the detection of disease-related biomarkers and environmental and organic hazards. They have also found widespread interest in the last years for the detection of waterborne and foodborne pathogens due to their label free character and high sensitivity. This Review is focused on the current electrochemical-based microorganism recognition approaches and putting them into context of other sensing devices for pathogens such as culturing the microorganism on agar plates and the polymer chain reaction (PCR) method, able to identify the DNA of the microorganism. Recent breakthroughs will be highlighted, including the utilization of microfluidic devices and immunomagnetic separation for multiple pathogen analysis in a single device. We will conclude with some perspectives and outlooks to better understand shortcomings. Indeed, there is currently no adequate solution that allows the selective and sensitive binding to a specific microorganism, that is fast in detection and screening, cheap to implement, and able to be conceptualized for a wide range of biologically relevant targets.

High capture efficiency of lectin surfaces for Cryptosporidium parvum biosensors

Detection of waterborne pathogens, such as Cryptosporidium, is essential for ensuring drinking water safety. Biosensors are one emerging technology aiming to improve the existing time-consuming and expensive monitoring approach. Surface functionalisation is a key aspect of biosensor development and here we propose that lectins offer an excellent alternative to antibodies offering a higher capture efficiency for oocysts. We also demonstrate that lectin immobilisation using a protocol of 11-mercaptoundecanoic acid followed by EDC-sulfo-NHS results in superior performance, compared to three other common surface functionalisation protocols, achieving an 86% capture efficiency. We also determine that capture efficiency with lectin surfaces is enhanced by the use of static conditions, in contrast to previous work with antibodies where surface capture was improved by convective conditions. These results will be highly useful in guiding the development of new biosensor systems for protozoan pathogen detection.

Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream.

Fecal indicator bacteria (FIB) are the basis for water quality regulations and are considered proxies for waterborne pathogens when conducting human health risk assessments. The direct detection of pathogens in water and simultaneous identification of the source of fecal contamination are possible with microarrays, circumventing the drawbacks to FIB approaches. A multigene target microarray was used to assess the prevalence of waterborne pathogens in a fecally impaired mixed-use watershed. The results indicate that fecal coliforms have improved substantially in the watershed since its listing as a 303(d) impaired stream in 2002 and are now near United States recreational water criterion standards. However, waterborne pathogens are still prevalent in the watershed, as viruses (bocavirus, hepatitis E and A viruses, norovirus, and enterovirus G), bacteria (Campylobacter spp., Clostridium spp., enterohemorrhagic and enterotoxigenic Escherichia coli, uropathogenic E. coli, Enterococcus faecalis, Helicobacter spp., Salmonella spp., and Vibrio spp.), and eukaryotes (Acanthamoeba spp., Entamoeba histolytica, and Naegleria fowleri) were detected. A comparison of the stream microbial ecology with that of sewage, cattle, and swine fecal samples revealed that human sources of fecal contamination dominate in the watershed. The methodology presented is applicable to a wide range of impaired streams for the identification of human health risk due to waterborne pathogens and for the identification of areas for remediation efforts.IMPORTANCE The direct detection of waterborne pathogens in water overcomes many of the limitations of the fecal indicator paradigm. Furthermore, the identification of the source of fecal impairment aids in identifying areas for remediation efforts. Multitarget gene microarrays are shown to simultaneously identify waterborne pathogens and aid in determining the sources of impairment, enabling further focused investigations. This study shows the use of this methodology in a historically impaired watershed in which total maximum daily load reductions have been successfully implemented to reduce risk. The results suggest that while the fecal indicators have been reduced more than 96% and are nearing recreational water criterion levels, pathogens are still detectable in the watershed. Microbial source tracking results show that additional remediation efforts are needed to reduce the impact of human sewage in the watershed.

Emerging biosensor platforms for the assessment of water-borne pathogens.

Pathogens are key contaminants in water that are responsible for the generation of various water-borne diseases, and include viruses, fungi, bacteria, and protozoan parasites. The pathogenic effects of these species in water depend on their shape, size, composition, and structure. The resulting water-borne diseases are a serious threat to the environment, including to humans and animals, and are directly responsible for environmental deterioration and pollution. The potential presence of these pathogens requires sensitive, powerful, efficient, and ideally real-time monitoring methods for their reproducible quantification. Conventional methods for pathogen detection mainly rely on time-consuming enrichment steps followed by biochemical identification strategies, which require assay times ranging from 24 h to up to a week. However, in recent years, significant efforts have been made towards the development of biosensing technologies enabling rapid and close-to-real-time detection of water-borne pathogens. This review summarizes recent developments in biosensors and sensing systems based on a variety of transducer technologies for water-quality monitoring, with specific focus on rapid pathogen detection.

A novel microfluidic module for rapid detection of airborne and waterborne pathogens

Abstract Different microfluidic design or function modules are the key for the microfluidic chip to fullfill various functions. We herein report a simple microfilter module that conveniently integrates semiporous membranes into polydimethylsiloxane (PDMS) substrates with leakage-free bonding, which achieves the efficient enrichment of airborne and waterborne pathogens on microfluidic chips. The microfilter is able to rapidly enrich and concentrate ∼10−1 μm-scale particles with high efficiency due to the complementary advantages of microfluidics (microliter-scale fluid control) and the nature of polyester (or polycarbonate) membranes (high precision and density of pores, and high hydrophilicity). Fabrication can be conveniently performed by effective loading control of the sealing agent. For further studies on different pathogen concentrations and detections, the module was integrated with loop-mediated isothermal amplification (LAMP) on a PDMS chip for enrichment and visual analysis of pathogens from body fluid samples with limited volume and low concentration. It was also capable of collection and in-situ immunofluorescence detection of parasite oocysts (cysts) in drinking water with recovery rates of ∼90%. Besides, by integration of the microfilter and a LAMP chip on a portable device, we successfully established a point-of-care system for enrichment and analysis of airborne pathogens.

Immunomagnetic separation for MEMS-based biosensor of waterborne pathogens detection

Rapid isolation and detection of special pathogens present in environmental drinking water is critical for water quality monitoring. Numerical analysis and experimental investigations on immunomagnetic capture and isolation of waterborne pathogens with magnetic nanoparticles (MNPs) in microfluidic channel are performed. A finite-element COMSOL-based model is established to demonstrate the novel method of on-chip capturing pathogens using MNPs together with periodic pulse magnetic field. Simulation results determine the optimum magnetic pole current and switching frequency for magnetic separation. With the magnetic isolation experiment platform built up, as a pathogen example of Escherichia coli O157:H7, the performance of the method is experimentally verified. Both numerical and experimental results are found to agree reasonably well. Results of these investigations show that the capture efficiency of the immunomagnetic separation method is more than 92%, which could be encouraging for the design and optimization of MEMS-based biosensor of waterborne pathogen detection.

Designing Biosensor for Detection of Water Borne Pathogens

Designing Biosensor for Detection of Water Borne Pathogens

Comparison of supervised machine learning algorithms for waterborne pathogen detection using mobile phone fluorescence microscopy

AbstractGiardia lambliais a waterborne parasite that affects millions of people every year worldwide, causing a diarrheal illness known as giardiasis. Timely detection of the presence of the cysts of this parasite in drinking water is important to prevent the spread of the disease, especially in resource-limited settings. Here we provide extended experimental testing and evaluation of the performance and repeatability of a field-portable and cost-effective microscopy platform for automated detection and counting ofGiardiacysts in water samples, including tap water, non-potable water, and pond water. This compact platform is based on our previous work, and is composed of a smartphone-based fluorescence microscope, a disposable sample processing cassette, and a custom-developed smartphone application. Our mobile phone microscope has a large field of view of ~0.8 cm2and weighs only ~180 g, excluding the phone. A custom-developed smartphone application provides a user-friendly graphical interface, guiding the users to capture a fluorescence image of the sample filter membrane and analyze it automatically at our servers using an image processing algorithm and training data, consisting of &gt;30,000 images of cysts and &gt;100,000 images of other fluorescent particles that are captured, including, e.g. dust. The total time that it takes from sample preparation to automated cyst counting is less than an hour for each 10 ml of water sample that is tested. We compared the sensitivity and the specificity of our platform using multiple supervised classification models, including support vector machines and nearest neighbors, and demonstrated that a bootstrap aggregating (i.e. bagging) approach using raw image file format provides the best performance for automated detection ofGiardiacysts. We evaluated the performance of this machine learning enabled pathogen detection device with water samples taken from different sources (e.g. tap water, non-potable water, pond water) and achieved a limit of detection of 12 cysts per 10 ml, an average cyst capture efficiency of ~79%, and an accuracy of ~95%. Providing rapid detection and quantification of waterborne pathogens without the need for a microbiology expert, this field-portable imaging and sensing platform running on a smartphone could be very useful for water quality monitoring in resource-limited settings.

Rapid Waterborne Pathogen Detection with Mobile Electronics.

Pathogen detection in water samples, without complex and time consuming procedures such as fluorescent-labeling or culture-based incubation, is essential to public safety. We propose an immunoagglutination-based protocol together with the microfluidic device to quantify pathogen levels directly from water samples. Utilizing ubiquitous complementary metal–oxide–semiconductor (CMOS) imagers from mobile electronics, a low-cost and one-step reaction detection protocol is developed to enable field detection for waterborne pathogens. 10 mL of pathogen-containing water samples was processed using the developed protocol including filtration enrichment, immune-reaction detection and imaging processing. The limit of detection of 10 E. coli O157:H7 cells/10 mL has been demonstrated within 10 min of turnaround time. The protocol can readily be integrated into a mobile electronics such as smartphones for rapid and reproducible field detection of waterborne pathogens.

An overview of dissolved organic carbon in groundwater and implications for drinking water safety

Dissolved organic carbon (DOC) is composed of a diverse array of compounds, predominantly humic substances, and is a near ubiquitous component of natural groundwater, notwithstanding climatic extremes such as arid and hyper-arid settings. Despite being a frequently measured parameter of groundwater quality, the complexity of DOC composition and reaction behaviour means that links between concentration and human health risk are difficult to quantify and few examples are reported in the literature. Measured concentrations from natural/unpolluted groundwater are typically below 4 mg C/l, whilst concentrations above these levels generally indicate anthropogenic influences and/or contamination issues and can potentially compromise water safety. Treatment processes are effective at reducing DOC concentrations, but refractory humic substance reaction with chlorine during the disinfection process produces suspected carcinogenic disinfectant by-products (DBPs). However, despite engineered artificial recharge systems being commonly used to remove DOC from recycled treated wastewaters, little research has been conducted on the presence of DBPs in potable groundwater systems. In recent years, the capacity to measure the influence of organic matter on colloidal contaminants and its influence on the mobility of pathogenic microorganisms has aided understanding of transport processes in aquifers. Additionally, advances in polymerase chain reaction techniques used for the detection, identification, and quantification of waterborne pathogens, provide a method to confidently investigate the behaviour of DOC and its effect on contaminant transfer in aquifers. This paper provides a summary of DOC occurrence in groundwater bodies and associated issues capable of indirectly affecting human health.

Detection of extremely low concentration waterborne pathogen using a multiplexing self-referencing SERS microfluidic biosensor

BackgroundIt is challenging to achieve ultrasensitive and selective detection of waterborne pathogens at extremely low levels (i.e., single cell/mL) using conventional methods. Even with molecular methods such as ELISA or PCR, multi-enrichment steps are needed which are labor and cost intensive. In this study, we incorporated nano-dielectrophoretic microfluidic device with Surface enhanced Raman scattering (SERS) technique to build a novel portable biosensor for easy detection and characterization of Escherichia coli O157:H7 at high sensitivity level (single cell/mL).ResultsA multiplexing dual recognition SERS scheme was developed to achieve one-step target detection without the need to separate target-bound probes from unbound ones. With three different SERS-tagged molecular probes targeting different epitopes of the same pathogen being deployed simultaneously, detection of pathogen targets was achieved at single cell level with sub-species specificity that has not been reported before in single-step pathogen detection.ConclusionThe self-referencing protocol implements with a Nano-dielectrophoretic microfluidic device potentially can become an easy-to-use, field-deployable spectroscopic sensor for onsite detection of pathogenic microorganisms.

A Spike Cocktail Approach to Improve Microbial Performance Monitoring for Water Reuse

Water reuse, via either centralized treatment of traditional wastewater or decentralized treatment and on-site reuse, is becoming an increasingly important element of sustainable water management. Despite advances in waterborne pathogen detection methods, low and highly variable pathogen levels limit their utility for routine evaluation of health risks in water reuse systems. Therefore, there is a need to improve our understanding of the linkage between pathogens and more readily measured process indicators during treatment. This paper describes an approach for constructing spiking experiments to relate the behavior of viral, bacterial, and protozoan pathogens with relevant process indicators. General issues are reviewed, and the spiking protocol is applied as a case study example to improve microbial performance monitoring and health risk evaluation in a water reuse system. This approach provides a foundation for the development of novel approaches to improve real or near-real time performance monitoring of water recycling systems.

Static mode microfluidic cantilevers for detection of waterborne pathogens

This paper reports on the first demonstration of polymeric microfluidic cantilever sensors. Microcantilever sensors, magnetic beads, and microfluidic technology have been combined to create a polymer based biosensor. Using cheap materials like polyimide, a simple fabrication method has been developed to produce cantilevers with an embedded microfluidic channel. The advantage of this approach is that the addition of a microfluidic channel enables the analysis of smaller volumes and increases the capture efficiency in applications detecting rare analytes. As a proof of principle the system has been applied for the detection of the waterborne protozoan parasite Cryptosporidium, achieving sensitivity comparable to QCM, whereas a previous set-up without the microfluidic channel was unable to detect the parasite.

Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L−1 assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation.

Microarrays/DNA Chips for the Detection of Waterborne Pathogens.

DNA microarrays are useful for the simultaneous detection of microorganisms in water samples. Specific probes targeting waterborne pathogens are selected with bioinformatics tools, synthesized and spotted onto a DNA array. Here, the construction of a DNA chip for waterborne pathogen detection is described, including the processes of probe in silico selection, synthesis, validation, and data analysis.