Low Concentration Of Pathogens Research Articles

A Critical Review on the Identification of Pathogens by Employing Peptide-Based Electrochemical Biosensors.

In this era of emerging pathogenic diseases, prompt and accurate detection of pathogens is crucial. Disease diagnosis, environmental monitoring and food safety all rely heavily on the identification of pathogens. Peptide-based electrochemical sensors due to their rapid response times, specificity and sensitivity have emerged as promising tools in the identification of pathogens. This review emphasizes the importance of peptides in detection of pathogens and different peptide-based electrochemical biosensors for the detection of pathogens. Peptides offer several advantages including strong binding affinity to a diverse array of pathogens including bacteria, viruses and fungi, tunable specificity and simple synthesis. Peptide-based electrochemical sensors employ different electrochemical techniques such as differential pulse voltammetry (DPV), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), amperometry and linear sweep voltammetry (LSV). The efficacy of peptide-based biosensors in detecting low concentrations of pathogens is highlighted, demonstrating the promising applications of these biosensors in early diagnosis and real-time monitoring. In addition, the review also addresses the current challenges in the field such as peptide stability, sensor reproducibility and interference from complex biological matrices. This review suggests potential resolutions and avenues for progress such as the development of multiplexed detection systems that can concurrently identify multiple pathogens and developments in peptide design and sensor miniaturization. In summary, this review highlights the substantial advancements and potential possibilities of peptide-based electrochemical biosensors in the realm of pathogen detection, thereby facilitating the development of safer and more effective diagnostic tools.

Microbial drinking water monitoring now and in the future.

Over time, humanity has addressed microbial water contamination in various ways. Historically, individuals resorted to producing beer to combat the issue. Fast forward to the 19th century, and we witnessed a scientific approach by Robert Koch. His groundbreaking gelatine plating method aimed to identify and quantify bacteria, with a proposed limit of 100 colony-forming units per millilitre (CFU/mL) to avoid Cholera outbreaks. Despite considerable advancements in plating techniques through experimentation with media compositions and growth temperatures, the reliance on a century-old method for water safety remains the state-of-the-art. Even though most countries succeed in producing qualitative water at the end of the production centres, it is difficult to control, and guarantee, the same quality during distribution. Rather than focusing solely on specific sampling points, we propose a holistic examination of the entire water network to ensure comprehensive safety. Current practices leave room for uncertainties, especially given the low concentrations of pathogens. Innovative methods like flow cytometry and flow cytometric fingerprinting offer the ability to detect changes in the microbiome of drinking water. Additionally, molecular techniques and emerging sequencing technologies, such as third-generation sequencing (MinION), mark a significant leap forward, enhancing detection limits and emphasizing the identification of unwanted genes rather than the unwanted bacteria/microorganisms itself. Over the last decades, there has been the realization that the drinking water distribution networks are complex ecosystems that, beside bacteria, comprise of viruses, protozoans and even isopods. Sequencing techniques to find eukaryotic DNA are necessary to monitor the entire microbiome of the drinking water distribution network. Or will artificial intelligence, big data and machine learning prove to be the way to go for (microbial) drinking water monitoring? In essence, it is time to transcend century-old practices and embrace modern technologies to ensure the safety of our drinking water from production to consumption.

Exposure to entomopathogenic fungus and high larval density induce a strong immune response and life‐history costs in black soldier fly, a commercially important insect

AbstractPathogen infection and conspecific density may considerably affect key life‐history traits of organisms. For naturally aggregating species, even low concentrations of pathogens or high larval densities may have detrimental effects. However, the detailed influence of these factors, particularly their interaction effect, is often overlooked in ecological and life‐history studies. To investigate the effects of conspecific density and pathogen infection on life‐history traits, we explored the influence of larval density (1 and 5 larvae cm−2, i.e., low and high density, respectively) on phenoloxidase (PO) activity, body mass, and development time of Hermetia illucens L. (Diptera: Stratiomyidae) larvae in the presence of two strains of the entomopathogenic fungus (EPF) Beauveria bassiana (Bals.‐Criv.) Vuill. We observed higher PO activity in EPF‐treated larvae than in the untreated control and a pronounced difference in PO activity between the two EPF‐strain treatments. Larvae reared at high density and treated with EPF showed higher PO activity than untreated larvae at low density. The EPF‐treated larvae and larvae reared at high density had longer larval periods than untreated larvae and larvae reared at low density, respectively. Larvae reared at high density also achieved reduced prepupal and pupal masses compared to conspecifics at low density. Interestingly, untreated larvae only achieved higher prepupal and pupal masses at low density, whereas at high density, the pattern was reversed (treated individuals had higher pupal masses). Overall, our results demonstrate that high density and fungal pathogens both induce a higher immune response compared to low density and pathogen‐free environments, but this comes with a cost of a longer larval period and reduced body mass.

CRISPR/Cas12a-enhanced DNA nanomachine for multiple respiratory pathogens detection.

Respiratory infection caused by pathogens is among the most prevalent health issues affecting people worldwide. Accurate and rapid screening of respiratory pathogens is crucial for selecting appropriate treatments to control epidemics. However, it is often challenged by two aspects: first, the low concentration of pathogens in the early stages of infection; second, the difficulty of analyzing multiple pathogens. Herein, we report a mass spectrometry strategy combining the CRISPR/Cas12a system with DNA nanomachines for respiratory pathogens detection. Thanks to the high sensitivity of the CRISPR/Cas12a-enhanced DNA nanomachine and the multiple analysis of elemental mass spectrometry, the proposed method was successfully applied for clinical sample analysis with a low detection limit of 28 amol, 30 amol, and 38 amol for SARS-CoV-2, influenza A virus subtype H1N1, and Mycoplasma pneumoniae, respectively.

Biomolecular survey on the main infectious causes of abortion in sheep in the Italian regions of Latium and Tuscany.

Ruminants play an important role in economic sustenance in many developing countries. Abortion is one of the most important causes of economic losses in sheep livestock and, for this reason, it is very important to know, at an early stage, which pathogens caused abortion. The aim of the study is to obtain data about the distribution of abortifacient pathogens in the Italian regions of Latium and Tuscany, the awareness of the distribution of infectious agents causing abortion could allow the development of an appropriate vaccination and prophylaxis plan, to avoid major economic losses. 388 abortions were collected during the 2015-2018 period. Organs, tissues, and swabs were subjected to DNA extraction and then analyzed with commercial q-PCR kits for the detection of the most common abortion pathogens circulating in these geographical areas. The positivity in 148 abortions was 56% for Chlamydia abortus, 14% for Coxiella burnetii, 16% for Salmonella spp, 12% for Toxoplasma gondii, and 2% for Neospora caninum. Interesting results were obtained for cases of abortions with co-infection of abortion pathogens. Diagnosing the cause of abortion remains a multifaceted process that may also include non-infectious factors such as deficiencies and toxicities. Further research is needed also to assess the role of low pathogen concentrations and co-infections in the abortions of sheep.

Emerging Applications of Nanobiosensors in Pathogen Detection in Water and Food.

Food and waterborne illnesses are still a major concern in health and food safety areas. Every year, almost 0.42 million and 2.2 million deaths related to food and waterborne illness are reported worldwide, respectively. In foodborne pathogens, bacteria such as Salmonella, Shiga-toxin producer Escherichia coli, Campylobacter, and Listeria monocytogenes are considered to be high-concern pathogens. High-concern waterborne pathogens are Vibrio cholerae, leptospirosis, Schistosoma mansoni, and Schistosima japonicum, among others. Despite the major efforts of food and water quality control to monitor the presence of these pathogens of concern in these kinds of sources, foodborne and waterborne illness occurrence is still high globally. For these reasons, the development of novel and faster pathogen-detection methods applicable to real-time surveillance strategies are required. Methods based on biosensor devices have emerged as novel tools for faster detection of food and water pathogens, in contrast to traditional methods that are usually time-consuming and are unsuitable for large-scale monitoring. Biosensor devices can be summarized as devices that use biochemical reactions with a biorecognition section (isolated enzymes, antibodies, tissues, genetic materials, or aptamers) to detect pathogens. In most cases, biosensors are based on the correlation of electrical, thermal, or optical signals in the presence of pathogen biomarkers. The application of nano and molecular technologies allows the identification of pathogens in a faster and high-sensibility manner, at extremely low-pathogen concentrations. In fact, the integration of gold, silver, iron, and magnetic nanoparticles (NP) in biosensors has demonstrated an improvement in their detection functionality. The present review summarizes the principal application of nanomaterials and biosensor-based devices for the detection of pathogens in food and water samples. Additionally, it highlights the improvement of biosensor devices through nanomaterials. Nanomaterials offer unique advantages for pathogen detection. The nanoscale and high specific surface area allows for more effective interaction with pathogenic agents, enhancing the sensitivity and selectivity of the biosensors. Finally, biosensors' capability to functionalize with specific molecules such as antibodies or nucleic acids facilitates the specific detection of the target pathogens.

A method of recovering the very low concentration of pathogens in river water by combining centrifugation and membrane filtration

Waterborne pathogens present major public health concerns because of the associated high mortality, morbidity and cost of treatment. Consumption of and contact with water contaminated by faeces is a significant risk factor for transmitting these organisms to humans. Their detection in a water sample is critical to ascertain potential risks to humans. They are relatively low in concentrations in surface waters, making their detection a challenge. Campylobacter is targeted here because it is one of the leading causes of enteric diseases globally, and consensus on the superiority of centrifugation over filtration, and vice versa, to recover Campylobacter spp. from river water samples for detection, has yet to be. Therefore, for this study river water sample was processed by combining both methods in a single set-up to concentrate Campylobacter spp. cells from water samples. This method of combining centrifugation and filtration can be expanded to other bacterial waterborne pathogens of public health importance.•Concentrating cells by centrifugation (14,000 × g for 30 min) to collect the pellets, followed by membrane filtration (using 0.45 µm) of the supernatant to trap any remaining suspended cells, and then pooling both pellet and residue presents an effective method for obtaining a satisfactory quantitative recovery of waterborne pathogens, such as Campylobacter spp. from environmental waters.•This is a critical need for quantitative microbial risk assessment studies.

Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater.

Detecting and identifying the origins of foodborne pathogen outbreaks is a challenging. The Next-Generation Sequencing (NGS) panel method offers a potential solution by enabling efficient screening and identification of various bacteria in one reaction. In this study, new NGS panel primer sets that target 18 specific virulence factor genes from six target pathogens (Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) were developed and optimized. The primer sets were validated for specificity and selectivity through singleplex PCR, confirming the expected amplicon size. Crosscheck and multiplex PCR showed no interference in the primer set or pathogenic DNA mixture. The NGS panel analysis of spiked water samples detected all 18 target genes in a single reaction, with pathogen concentrations ranging from 108 to 105 colony-forming units (CFUs) per target pathogen. Notably, the total sequence read counts from the virulence factor genes showed a positive association with the CFUs per target pathogen. However, the method exhibited relatively low sensitivity and occasional false positive results at low pathogen concentrations of 105 CFUs. To validate the detection and identification results, two sets of quantitative real-time PCR (qPCR) analyses were independently performed on the same spiked water samples, yielding almost the same efficiency and specificity compared to the NGS panel analysis. Comparative statistical analysis and Spearman correlation analysis further supported the similarity of the results by showing a negative association between the NGS panel sequence read counts and qPCR cycle threshold (Ct) values. To enhance NGS panel analysis for better detection, optimization of primer sets and real-time NGS sequencing technology are essential. Nonetheless, this study provides valuable insights into applying NGS panel analysis for multiple foodborne pathogen detection, emphasizing its potential in ensuring food safety.

Establishment of a Fast Diagnostic Method for Sepsis Pathogens Based on M1 Bead Enrichment

Blood culture-based sepsis diagnostic methods usually cannot obtain positive results in a timely manner. Molecular diagnostic methods, such as real-time PCR without blood culture, would be more time-saving and suitable for pathogenic diagnosis of sepsis, while their sensitivities have always been unsatisfactory for the usually low concentration of pathogens in the blood of sepsis patients. In this study, we established a fast diagnostic method using magnetic beads coated with human recombined mannose-binding lectin that makes it possible to concentrate pathogens from human plasma that have low concentrations of pathogens. With subsequent microculture (MC) and real-time PCR, this method allowed the detection of 1-10 CFUs/ml of Staphylococcus aureus, Group A Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Candida tropicalis, or C. albicans from human plasma within 9.5h, which was 21-80h earlier than blood culture. The combination of pathogen enrichment and MC made the detection of sepsis-causing pathogens more time-saving and more sensitive than blood culture or real-time PCR alone.

On-site and dual-mode detection of live Vibrio parahaemolyticus in waters: A universal pathogen sensing platform based on a smart hydrogel aptasensor imbedded with gold nanoclusters

Usually, a low concentration of pathogens proliferate rapidly in waters to cause serious threats to human health. It is necessary to develop on-site and speedy assays to detect them. In this work, a smart hydrogel aptasensor was assembled in Eppendorf tubes for quantifying Vibrio parahaemolyticus (V.P). It employed a dual-mode detection strategy combining visual fluorescence (FL) and microfluidic chip (MC) methods. In this aptasensor, the hydrogel contained ATP aptamer together with gold nanoclusters (AuNCs) as signal tags, and V.P aptamer was modified on tube lids. Firstly, trace V.P in waters was specifically enriched by the lid and then hydrolyzed to release ATP which was combined with ATP aptamer in hydrogel to form ATP-aptamer complex. The complex together with AuNCs was subsequently released into the supernatant for dual-mode detection. ATP-aptamer was detected by MC and AuNCs by FL. By the assay, as low as 100 CFU·mL−1 V.P was determined by FL within 45 min and 10 CFU·mL−1 by MC. The dual-mode sensing platform exhibits the following advantages: Firstly, live pathogens were quantitatively and on-site detected. Secondly, the lid’s enrichment of V.P and signal transduction from one V.P to 1014 ATP could double-amplify signals and increase the detection efficiency. Finally, the samples quickly screened by visual FL on-site, and then accurately quantified by MC in the laboratory, which greatly shortens the detection period. It has been successfully utilized for on-site detecting V.P in aquatic waters and could be extended to other bacteria measurements through changing the relative aptamer on the tube lid.

Inverse design of a single-frequency diffractive biosensor based on the reporter cleavage detection mechanism.

Vertically interrogated porous silicon (PSi) interferometric biosensors have shown high potential for sensing bio-molecules as they combine high detection sensitivity with simplicity of fabrication, functionalization, optical coupling, and interfacing with microfluidic systems. However, most interferometric sensor designs require either broadband or wavelength-tunable light sources as well as wide-angle detection schemes, increasing their complexity and cost for point-of-care biosensing applications. The limit of detection of such sensors is also constrained by the small size and low refractive index of biological molecules, making it hard to detect very low concentrations of pathogens. In this work, we use a large-scale computational "inverse design" technique to demonstrate a single-frequency, fixed-angle PSi-based biosensor, which exploits a recently developed high-contrast reporter cleavage detection (HCCD) technique. The HCCD sensors detect high-index reporter cleavage events instead of low-index target analyte capture events as typical for traditional label-free optical biosensors. We use the inverse design approach to discover an optimal configuration of a PSi biosensor that makes use of the extended achievable range of cleavage-induced PSi effective index variations and can be interrogated at a single frequency and at a fixed angle. The optimized design in the form of a one-dimensional PSi grating exhibits the change in the reflectance up to 55 % at the interrogation angle of 12∘ and wavelength of 600 nm, which is caused by cleavage of Au nanoparticle reporters initially occupying 2% of the sensor surface area. The maximum possible change in reflectance is predicted to be 222 % (for a two-dimensional freeform design not amenable to fabrication). This demonstration may pave the way for developing new or redesigned conventional interferometric and colorimetric point-of-care biosensor systems in combination with the cleavage-based detection schemes.

Integrated Photonic System for Early Warning of Cyanobacterial Blooms in Aquaponics.

Cyanobacterial blooms produce hazardous toxins, deplete oxygen, and secrete compounds that confer undesirable organoleptic properties to water. To prevent bloom appearance, the World Health Organization has established an alert level between 500 and 2000 cells·mL-1, beyond the capabilities of most optical sensors detecting the cyanobacteria fluorescent pigments. Flow cytometry, cell culturing, and microscopy may reach these detection limits, but they involve both bulky and expensive laboratory equipment or long and tedious protocols. Thus, no current technology allows fast, sensitive, and in situ detection of cyanobacteria. Here, we present a simple, user-friendly, low-cost, and portable photonic system for in situ detection of low cyanobacterial concentrations in water samples. The system integrates high-performance preconcentration elements and optical components for fluorescence measurement of specific cyanobacterial pigments, that is, phycocyanin. Phycocyanin has demonstrated to be more selective to cyanobacteria than other pigments, such as chlorophyll-a, and to present an excellent linear correlation with bacterial concentration from 102 to 104 cell·mL-1 (R2 = 0.99). Additionally, the high performance of the preconcentration system leads to detection limits below 435 cells·mL-1 after 10 min in aquaponic water samples. Due to its simplicity, compactness, and sensitivity, we envision the current technology as a powerful tool for early warning and detection of low pathogen concentrations in water samples.

Rapid and sensitive detection of Salmonella spp. in raw minced meat samples using droplet digital PCR

With its ability to easily infect each step of food production chain, Salmonella spp. stands as one of the most important foodborne pathogens which impose threats against economy and human health by creating high morbidity and mortality, worldwide. Although sensitive screening and detection methodologies based on common conventional techniques have been developed for Salmonella spp., their long turnaround time and strict legislative demands directs the researchers to develop more sensitive and rapid methods. Digital PCR has many potential advantages over its closest competitor, Real-Time PCR. It gives more accurate, sensitive, and precise results, especially for samples that contain low pathogen concentrations and shows more resistance against PCR inhibitors. In this study, we developed a droplet digital PCR screening methodology with a short pre-enrichment step. For this purpose, 25 g raw minced meat was subjected to Salmonella contamination, and following a very short 3 h enrichment step, bacterial isolation from 5 mL of sample was achieved. For the specific detection of the Salmonella spp., bipA gene region was selected as a better candidate. Subsequently, droplet digital PCR setup was performed and detection sensitivity of the test was determined as 1.39 cfu/mL. Furthermore, parallel experiments were also conducted using Real-Time PCR, for comparative and validative purposes. Conclusively, data gathered in this study have shown that droplet digital PCR with our proposed setup can sensitively detect Salmonella spp. in raw minced meat samples while generating the same-day results.

Microcantilever Based Dual Mode Biosensor for Agricultural Applications

With the aim of increasing crop yields, guiding precision farming applications and for more sustainable disease management practices development of early diagnostics sensors for pathogens in agriculture is vital. A sensitive method of diagnosis which will detect the low concentration of pathogens in the initial stages of infection will allow early diagnosis and help in saving the crop. Microcantilever based biosensors are ideal devices for such kind of applications. This paper presents development of a highly sensitive dual mode operated sensor for agricultural pathogen detection. Micromolar concentartion of Ralstonia solanacearum antigen in liquid medium was detected optically by using the ultrasensitive Mecwins® SCALA platform while electrically using a Keithley source meter. Agricultural pathogen detection employing optical as well as electrical readout using a piezoresistive microcantilever platform in both static and dynamic modes of operation is reported here for the first time.

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.

Pathogen Concentration Combined Solid-Phase PCR on Supercritical Angle Fluorescence Microlens Array for Multiplexed Detection of Invasive Nontyphoidal Salmonella Serovars.

Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella-spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S. typhimurium and S. enteritidis, respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

Development of highly efficient protocols for extraction and amplification of cytomegalovirus DNA from dried blood spots for detection and genotyping of polymorphic immunomodulatory genes.

Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection.

Particle Diffusometry: An Optical Detection Method for Vibrio cholerae Presence in Environmental Water Samples

There is a need for a rapid, robust, and sensitive biosensor to identify low concentrations of pathogens in their native sample matrix without enrichment or purification. Nucleic acid-based detection methods are widely accepted as the gold standard in diagnostics, but robust detection of low concentrations of pathogens remains challenging. Amplified nucleic acids produce more viscous solutions, which can be measured by combining these products with fluorescent particles and measuring the change in the particle diffusion coefficient using a technique known as particle diffusometry. Here, we utilize Vibrio cholerae (V. cholerae) as a proof-of-concept for our detection system due to its inherently low concentration in environmental water samples. We demonstrate that particle diffusometry can be used to detect down to 1 V. cholerae cell in molecular-grade water in 20 minutes and 10 V. cholerae cells in pond water in just 35 minutes in 25 µL reaction volumes. The detection limit in pond water is environmentally relevant and does not require any enrichment or sample preparation steps. Particle diffusometry is 10-fold more sensitive than current gold standard fluorescence detection of nucleic acid amplification. Therefore, this novel measurement technique is a promising approach to detect low levels of pathogens in their native environments.

Simple and label-free pathogen enrichment via homobifunctional imidoesters using a microfluidic (SLIM) system for ultrasensitive pathogen detection in various clinical specimens

Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.

A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment

Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5–10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.