New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

Automated liquid handling robot for rapid lateral flow assay development.

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.

Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests.

The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/µL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.

SARS-CoV-2 Coronavirus Nucleocapsid Antigen-Detecting Half-Strip Lateral Flow Assay Toward the Development of Point of Care Tests Using Commercially Available Reagents.

The SARS-CoV-2 pandemic has created an unprecedented need for rapid diagnostic testing to enable the efficient treatment and mitigation of COVID-19. The primary diagnostic tool currently employed is reverse transcription polymerase chain reaction (RT-PCR), which can have good sensitivity and excellent specificity. Unfortunately, implementation costs and logistical problems with reagents during the global SARS-CoV-2 pandemic have hindered its universal on demand adoption. Lateral flow assays (LFAs) represent a class of diagnostic that, if sufficiently clinically sensitive, may fill many of the gaps in the current RT-PCR testing regime, especially in low- and middle-income countries (LMICs). To date, many serology LFAs have been developed, though none meet the performance requirements necessary for diagnostic use cases, primarily due to the relatively long delay between infection and seroconversion. However, on the basis of previously reported results from SARS-CoV-1, antigen-based SARS-CoV-2 assays may have significantly better clinical sensitivity than serology assays. To date, only a very small number of antigen-detecting LFAs have been developed. Development of a half-strip LFA is a useful first step in the development of any LFA format. In this work, we present a half-strip LFA using commercially available antibodies for the detection of SARS-CoV-2. We have tested this LFA in buffer and measured an LOD of 0.65 ng/mL (95% CI of 0.53 to 0.77 ng/mL) ng/mL with recombinant antigen using an optical reader with sensitivity equivalent to a visual read. Further development, including evaluating the appropriate sample matrix, will be required for this assay approach to be made useful in a point of care setting, though this half-strip LFA may serve as a useful starting point for others developing similar tests.

Wash-Free, Digital Immunoassay in Polydisperse Droplets.

Immunoassays are important for the detection of proteins to enable disease identification and monitor treatment, but many immunoassays suffer from sensitivity limitations. The development of digital assays has enabled highly sensitive biomarker detection and quantification, but the necessary devices typically require precisely controlled volumes to reduce biases in concentration estimates from compartment size variation. These constraints have led to systems that are often expensive, cumbersome, and challenging to operate, confining many digital assays to centralized laboratories. To overcome these limitations, we have developed a simplified digital immunoassay performed in polydisperse droplets that are prepared without any specialized equipment. This polydisperse digital droplet immunoassay (ddIA) uses proximity ligation to remove the need for wash steps and simplifies the system to a single reagent addition step. Using interleukin-8 (IL-8) as an example analyte, we demonstrated the concept with samples in buffer and diluted whole blood with limits of detection of 0.793 pM and 1.54 pM, respectively. The development of a one-pot, washless assay greatly improves usability compared to traditional immunoassays or digital-based systems that rely heavily on wash steps and can be run with common and readily available laboratory equipment such as a heater and simple fluorescent microscope. We also developed a stochastic model with physically meaningful parameters that can be utilized to optimize the assay and enable quantification without standard curves, after initial characterization of the parameters. Our polydisperse ddIA assay serves as an example of sensitive, lower-cost, and simpler immunoassays suitable for both laboratory and point-of-care applications.

General methods for quantitative interpretation of results of digital variable-volume assays.

In digital assays, devices are typically considered to require precisely controlled volumes since variation in compartment volumes causes biases in concentration estimates. To enable more possibilities in device design, we derived two methods to accurately calculate target concentrations from raw results when the compartment volume may vary and may not follow known parametrically described distributions. The Digital Variable Volume (dvv) method uses volumes of ON compartments (those with positive signals) and the total sample volume, while the Digital Variable Volume Approximation (dvva) method uses the number of ON compartments, the total number of compartments, and a set of separately measured volumes. We verified the trueness of the dvv and dvva methods using simulated assays where volumes followed an empirical distribution (based on measured droplet volumes) and well known distributions with a wide range of standard deviations. We applied both methods to digital PCR experiments with polydisperse volumes, and also derived equations to estimate standard errors and limits of detection. The dvv method allows the compartment volume to follow any distribution in each assay run, the dvva method allows for quantification without in-assay volume measurements, and both methods potentially enable new designs of digital assays.

Simple Polydisperse Droplet Emulsion Polymerase Chain Reaction with Statistical Volumetric Correction Compared with Microfluidic Droplet Digital Polymerase Chain Reaction.

Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.

Threshold-Based Quantification in a Multiline Lateral Flow Assay via Computationally Designed Capture Efficiency.

Lateral flow assays (LFAs) are widely used for yes/no detection of analytes, but they are not well-suited for quantification. We show that the sensitivity of the test line in a lateral flow assay can be tuned to appear at a specific sample concentration by varying the density of capture molecules at the test line and that when test lines tuned for different responses are combined into a single test strip, lines appear at specific thresholds of sample concentration. We also developed a model based on mass-action kinetics that accurately described test line signal and shape over a wide matrix of capture molecules and sample concentrations in single-line strips. The model was used to design a three-line test strip with lines designed to appear at logarithmically spaced sample concentrations, and the experiments showed a remarkable match to predictions. The response of this "graded ladder bar" format is due to the effect of test line concentration on capture efficiency at each test line, not on sample depletion effects, and the effect is maintained whether a system is under kinetic or equilibrium control. These features enable design of nonlinear responses (logarithmic here) and suggest robustness for different systems. Thus, the graded ladder bar format could be a useful tool for applications requiring quantification of sample concentrations over a wide dynamic range.

Polydisperse emulsion digital assay to enhance time to detection and extend dynamic range in bacterial cultures enabled by a statistical framework.

Microbiological culture remains the most sensitive method for detecting viable and infectious bacteria, but these methods often require at least 24 hours to visibly identify bacterial growth. Lab-on-a-chip applications have utilized methods to isolate bacteria in picoliter-sized reaction vessels, resulting in digitized signals that offer improved time-to-detection and improved quantification. Although a great improvement, these approaches typically require expensive and specialized equipment, trained laboratory personnel, and maximum addressable volumes that can be orders of magnitude less than needed for clinically relevant limits of detection. To address these limitations, we have developed a simple method for preparing and semi-quantitatively analyzing small-volume droplets for performing digital culture, allowing for the detection of bacteria. This work includes a description of the method, characterization of resulting droplet sizes, comparison to traditional culture, and a statistical framework to quantify results. Though polydisperse, the droplet size distribution was consistent over different experiments, and there was a correlation between the observed number of positive droplets and the bulk concentration that can serve as a calibration curve for samples with unknown droplet size distributions. This statistical framework enables the simplification of droplet preparation and allows for accurate quantification even with polydisperse droplet sizes. The application of this method can also be extended to a variety of settings for the detection or quantification of bacteria in complex samples.

Towards biomarker-based tests that can facilitate decisions about prevention and management of preeclampsia in low-resource settings.

In recent years, an increasing amount of literature is emerging on candidate urine and blood-based biomarkers associated with incidence and severity of preeclampsia (PE) in pregnant women. While enthusiasm on the usefulness of several of these markers in predicting PE is evolving, essentially all work so far has focused on the needs of high-resource settings and high-income countries, resulting primarily in multi-parameter laboratory assays based on proteomic and metabolomics analysis techniques. These highly complex methods, however, require laboratory capabilities that are rarely available or affordable in low-resource settings (LRS). The importance of quantifying maternal and perinatal risks and identifying which pregnancies can be safely prolonged is also much greater in LRS, where intensive care facilities that can rapidly respond to PE-related health threats for women and infants are limited. For these reasons, simple, low cost, sensitive, and specific point-of-care (POC) tests are needed that can be performed by antenatal health care providers in LRS and that can facilitate decisions about detection and management of PE. Our study aims to provide a comprehensive systematic review of current and emerging blood and urine biomarkers for PE, not only on the basis of their clinical performance, but also of their suitability to be used in LRS-compatible test formats, such as lateral flow and other variants of POC rapid assays.

Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.

We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.

New manual qPCR assay validated on tongue swabs collected and processed in Uganda shows sensitivity that rivals sputum-based molecular TB diagnostics.

Background: Reliance on sputum-based testing is a key barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce and sputum processing increases the complexity and cost of molecular assays. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. Methods: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at two health centers in Kampala, Uganda. We collected routine demographic and clinical information, sputum for routine TB testing (one Xpert MTB/RIF Ultra® and two liquid cultures), and up to four tongue swabs for same-day qPCR. We evaluated tongue swab qPCR diagnostic accuracy in reference to sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared two swab types (Copan FLOQSWAB® and Steripack® spun polyester swabs). Results: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were living with HIV (PLHIV) and 32.3% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% [96.2-99.1] of participants. Tongue swab qPCR sensitivity was 91.0% [84.6-94.9] and specificity 98.9% [96.2-99.8] vs. microbiological reference standard (MRS). A single tongue swab recovered a seven-log range of TB copies, with a decreasing recovery trend among four serial swabs. We found no difference between swab types. Conclusions: Tongue swabs show promise as an alternative to sputum for TB diagnosis, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

Microfluidic diagnostic technologies for global public health.

The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.

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.

A Novel Malaria Lateral Flow Assay for Detecting Plasmodium falciparum Lactate Dehydrogenase in Busia, Uganda.

Rapid diagnostic tests (RDTs) for Plasmodium falciparum commonly detect histidine-rich protein 2 (HRP-2), but HRP-2 deletions are increasingly recognized. We evaluated a prototype test detecting parasite lactate dehydrogenase (pLDH) and compared it to commercially available RDTs at a health facility in Uganda, using quantitative polymerase chain reaction as a gold standard. The prototype pLDH test had a high sensitivity for infections with at least 100 parasites/µL (98%), comparable to HRP-2, and greater than an existing pLDH RDT (89%). Specificity for the prototype test was 99.5%, which is greater than the HRP-2 tests (93-95%). Therefore, the prototype pLDH test may be an attractive alternative malaria diagnostic.

Association of elevated E6 oncoprotein with grade of cervical neoplasia using PDZ interaction-mediated precipitation of E6.

OBJECTIVE: To determine the expression of human papillomavirus (HPV) type 16 E6 oncoprotein in cervical specimens of women with and without cervical intraepithelial neoplasia (CIN). MATERIALS AND METHODS: Cervical specimens from 2,530 unscreened women aged 30 to 54 years from Shanxi, China, were obtained. All women were assessed by liquid-based cytology, high-risk HPV DNA tests, and colposcopy with directed biopsy and endocervical curettage as necessary. Women with abnormal cytologic results or positive HPV DNA results were recalled for colposcopy, 4-quadrant cervical biopsies, and endocervical curettage. Women with biopsy-proven CIN and cancer and a convenience sample of HC2-positive, disease-negative women were tested for the presence of HPV-16 infection via HPV-16 E6 DNA-specific polymerase chain reaction. A PDZ interaction-mediated E6 oncoprotein precipitation method followed by E6-specific Western blot was performed on specimens from women with HPV-16 infections. Associations between elevated expression of E6 oncoprotein and CIN 2 and 3 were determined using logistic regression and a reference category of CIN 1 and disease-negative. RESULTS: A significant trend for the detection of HPV-16 E6 oncoprotein in specimen of women with proven HPV-16 infection was determined: 0% (0/12), 12.5% (1/8), 36.4% (4/11), and 42.9% (3/7) of those with negative findings, CIN 1, 2, and 3, respectively (p = .01). Compared with the category combining negative findings and CIN 1, detection of E6 oncoprotein was associated with CIN 2 (odds ratio = 10.9, p = .05) and CIN 3 (odds ratio = 14.3, p = .04). CONCLUSIONS: There is a significant association between elevated expression of E6 oncoprotein and grade of CIN. This finding seems consistent with the role played by E6 oncoprotein in carcinogenesis.

Laboratory operations, specimen processing, and handling for viral load testing and surveillance.

RNA remains the most informative and accurate biomarker for human immunodeficiency virus type 1 load diagnostics and for surveillance of drug resistance markers. Viral load testing by nucleic acid amplification currently is a complex and expensive test that is restricted to centralized laboratory testing. Successful extension of centralized viral load testing to rural or remote settings is a major challenge. Emerging nucleic acid-based technologies are progressing rapidly toward platforms appropriate for field use in low-resource settings, leaving a growing gap for sample processing technologies that complement them. One area in which new technologies could be applied to improve access is clinical specimen preservation and processing. Novel technologies that extract nucleic acid from clinical specimens and stabilize it at the point of specimen collection could fill this gap. In addition, these technologies may provide alternative viral load detection and surveillance solutions to the current centralized laboratory testing paradigm.

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.

Antibody Screening Results for Anti-Nucleocapsid Antibodies Toward the Development of a Lateral Flow Assay to Detect SARS-CoV-2 Nucleocapsid Protein.

The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.