A microfluidic device and instrument prototypes for the detection of Escherichia coli in water samples using a phage-based bioluminescence assay.

Current quantification methods of Escherichia coli (E. coli) contamination in water samples involve long incubation, laboratory equipment and facilities, or complex processes that require specialized training for accurate operation and interpretation. To address these limitations, we have developed a microfluidic device and portable instrument prototypes capable of performing a rapid and highly sensitive bacteriophage-based assay to detect E. coli cells with detection limit comparable to traditional methods in a fraction of the time. The microfluidic device combines membrane filtration and selective enrichment using T7-NanoLuc-CBM, a genetically engineered bacteriophage, to identify 4.1 E. coli CFU in 100 mL of drinking water within 5.5 hours. The microfluidic device was designed and tested to process up to 100 mL of real-world drinking water samples with turbidities below 10 NTU. Prototypes of custom instrumentation, compatible with our valveless microfluidic device and capable of performing all of the assay's units of operation with minimal user intervention, demonstrated similar assay performance to that obtained on the benchtop assay. This research is the first step towards a faster, portable, and semi-automated, phage-based microfluidic platform for improved in-field water quality monitoring in low-resource settings.

Rapid, sensitive, and low-cost detection of Escherichia coli bacteria in contaminated water samples using a phage-based assay.

Inadequate drinking water quality is among the major causes of preventable mortality, predominantly in young children. Identifying contaminated water sources remains a significant challenge, especially where resources are limited. The current methods for measuring Escherichia coli (E. coli), the WHO preferred indicator for measuring fecal contamination of water, involve overnight incubation and require specialized training. In 2016, UNICEF released a Target Product Profile (TPP) to incentivize product innovations to detect low levels of viable E. coli in water samples in the field in less than 6 h. Driven by this challenge, we developed a phage-based assay to detect and semi-quantify E. coli. We formulated a phage cocktail containing a total of 8 phages selected against an extensive bacterial strain library and recombined with the sensitive NanoLuc luciferase reporter. The assay was optimized to be processed in a microfluidic chip designed in-house and was tested against locally sourced sewage samples and on drinking water sources in Nairobi, Kenya. With this assay, combined with the microfluidic chip platform, we propose a complete automated solution to detect and semi-quantify E. coli at less than 10 MPN/100 mL in 5.5 h by minimally trained personnel.

A SARS-CoV-2 coronavirus nucleocapsid protein antigen-detecting lateral flow assay.

Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.

Clinical validation of an open-access SARS-COV-2 antigen detection lateral flow assay, compared to commercially available assays.

Rapid tests for SARS-COV-2 infection are important tools for pandemic control, but current rapid tests are based on proprietary designs and reagents. We report clinical validation results of an open-access lateral flow assay (OA-LFA) design using commercially available materials and reagents, along with RT-qPCR and commercially available comparators (BinaxNOW® and Sofia®). Adult patients with suspected COVID-19 based on clinical signs and symptoms, and with symptoms ≤7 days duration, underwent anterior nares (AN) sampling for the OA-LFA, Sofia®, BinaxNOW ™, and RT-qPCR, along with nasopharyngeal (NP) RT-qPCR. Results indicate a positive predictive agreement with NP sampling as 69% (60% -78%) OA-LFA, 74% (64% - 82%) Sofia®, and 82% (73% - 88%) BinaxNOW™. The implication for these results is that we provide an open-access LFA design that meets the minimum WHO target product profile for a rapid test, that virtually any diagnostic manufacturer could produce.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility.

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Genome Sequences of 38 Bacteriophages Infecting Escherichia coli, Isolated from Raw Sewage.

Here, we report the complete genome sequences of 38 novel bacteriophages infecting Escherichia coli, isolated from a raw sewage source in Washington. Of these phages, 26 are under 100 kb, 11 are near 170 kb, and 1 352-kb jumbo phage was discovered.

A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages.

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water.

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.

A paper microfluidic cartridge for automated staining of malaria parasites with an optically transparent microscopy window.

A paper microfluidic cartridge for the automated staining of malaria parasites (Plasmodium) with acridine orange prior to microscopy is presented. The cartridge enables simultaneous, sub-minute generation of both thin and thick smears of acridine orange stained parasites. Parasites are stained in a cellulose matrix, after which the parasites are ejected via capillary forces into an optically transparent chamber. The unique slanted design of the chamber ensures that a high percentage of the stained blood will be of the required thickness for a thin smear, without resorting to spacers or other methods that can increase production cost or require tight quality controls. A hydrophobic snorkel facilitates the removal of air bubbles during filling. The cartridge contains both a thin smear region, where a single layer of cells is presented unobstructed, for ease of species identification, and a thick smear region, containing multiple cell layers, for enhanced limit of detection.