No abstract

Each day, approximately 830 women and 7,400 newborns die from complications during pregnancy and childbirth. Improving maternal and neonatal health will require bringing rapid diagnosis and treatment to the point of care in low-resource settings. However, to date there are few diagnostic tools available that can be used at the point of care to detect the leading causes of maternal and neonatal mortality in low-resource settings. Here we review both commercially available diagnostics and technologies that are currently in development to detect the leading causes of maternal and neonatal mortality, highlighting key gaps in development where innovative design could increase access to technology and enable rapid diagnosis at the bedside.

Sickle cell disease (SCD) is a group of common, life-threatening disorders caused by a point mutation in the β globin gene. Early diagnosis through newborn and early childhood screening, parental education, and preventive treatments are known to reduce mortality. However, the cost and complexity of conventional diagnostic methods limit the feasibility of early diagnosis for SCD in resource-limited areas worldwide. Although several point-of-care tests are commercially available, most are antibody-based tests, which cannot be used in patients who have recently received a blood transfusion. Here, we describe the development of a rapid, low-cost nucleic acid test that uses real-time fluorescence to detect the point mutation encoding hemoglobin S (HbS) in one round of isothermal recombinase polymerase amplification (RPA). When tested with a set of clinical samples from SCD patients and healthy volunteers, our assay demonstrated 100% sensitivity for both the β A globin and β S globin alleles and 94.7 and 97.1% specificities for the β A globin allele and β S globin allele, respectively ( n = 91). Finally, we demonstrate proof-of-concept sample-to-answer genotyping of genomic DNA from capillary blood using an alkaline lysis procedure and direct input of diluted lysate into RPA. The workflow is performed in <30 min at a cost of <$5 USD on a commercially available benchtop fluorimeter and an open-source miniature fluorimeter. This study demonstrates the potential utility of a rapid, sample-to-answer nucleic acid test for SCD that may be implemented near the point of care and could be adapted to other disease-causing point mutations in genomic DNA.

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69–91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

Frequent and accessible testing is a critical tool to contain the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To develop low-cost rapid tests, many researchers have used reverse transcription loopmediated isothermal amplification (RT-LAMP) with fluorescent readout. Fluorescent LAMP-based assays can be performed using cost-effective, portable, isothermal instruments that are simpler to use and more rugged than polymerase chain reaction (PCR) instruments. However, false-positive results due to nonspecific priming and amplification have been reported for a number of LAMP-based assays. In this report, we implemented a RT-LAMP assay for SARS-CoV-2 on a portable isothermal fluorimeter and a traditional thermocycler; nonspecific amplification was not observed using the thermocycler but did occur frequently with the isothermal fluorimeter. We explored 4 strategies to optimize the SARS-CoV-2 RT-LAMP assay for use with an isothermal fluorimeter and found that overlaying the reaction with mineral oil and including the enzyme Tte UvrD helicase in the reaction eliminated the problem. We anticipate these results and strategies will be relevant for use with a wide range of portable isothermal instruments.

High-risk human papillomavirus (HPV) DNA testing is widely acknowledged as the most sensitive cervical cancer screening method but has limited availability in resource-limited settings, where the burden of cervical cancer is highest. Recently, HPV DNA tests have been developed for use in resource-limited settings, but they remain too costly for widespread use and require instruments that are often limited to centralized laboratories. To help meet the global need for low-cost cervical cancer screening, we developed a prototype, sample-to-answer, point-of-care test for HPV16 and HPV18 DNA. Our test relies on isothermal DNA amplification and lateral flow detection, two technologies that reduce the need for complex instrumentation. We integrated all test components into a low-cost, manufacturable platform, and performance of the integrated test was evaluated with synthetic samples, provider-collected clinical samples in a high-resource setting in the United States, and self-collected clinical samples in a low-resource setting in Mozambique. We demonstrated a clinically relevant limit of detection of 1000 HPV16 or HPV18 DNA copies per test. The test requires six user steps, yields results in 45 min, and can be performed using a benchtop instrument and minicentrifuge by minimally trained personnel. The projected per-test cost is <$5, and the projected instrumentation cost is <$1000. These results show the feasibility of a sample-to-answer, point-of-care HPV DNA test. With the inclusion of other HPV types, this test has the potential to fill a critical gap for decentralized and globally accessible cervical cancer screening.

Background The global COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid, accurate, and accessible diagnostics to enable timely treatment and outbreak control. However, current diagnostic tests based on RT-qPCR are insufficient to meet the global testing demand because of their high cost and complexity and supply chain shortages. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a promising alternative to RT-qPCR because of its sensitivity, speed, and robustness to sample inhibitors. Here, we describe the development and optimisation of a sample-to-answer workflow, including a simple lysis and inactivation protocol that provides results in <1h, using inexpensive and readily available reagents. Further, we assess the sensitivity and specificity of the developed RT-LAMP assay against a RT-qPCR. Methods We collected samples from asymptomatic healthcare workers at The University of Texas MD Anderson Cancer Center and inpatients at Lyndon B Johnson Hospital in Houston, TX, USA. Nasopharyngeal swabs were collected by medical providers and were placed directly into 300 µL of an optimised lysis buffer. Samples were heat inactivated at 95°C for 5 mins before direct amplification in a RT-LAMP assay using previously published primer sets. Heating and real-time monitoring was performed using a Bio-Rad CFX96 thermocycler and an Axxin T8-ISO, a benchtop fluorimeter designed for point-of-care settings. We compared results from the RT-LAMP test with standardof-care RT-qPCR results on paired nasopharyngeal swabs collected into Universal Viral Transport Media. Findings The developed RT-LAMP assay demonstrated a limit of detection of 4-5 virions/µL. The test requires a swab, two tubes, prepared lysis buffer, a heat block, pipettes, RT-LAMP reagents, and the real-time fluorimeter. Samples were collected between April 14, 2020 and Aug 12, 2020, and results from 74 enrolled participants were analysed in the optimised workflow. Thirty nine participants tested positive for SARS-CoV-2 and 35 participants tested negative in the hospital-administered RT-qPCR test. All 74 nasopharyngeal swab eluates were tested with our assay on the Bio-Rad CFX96; 72 nasopharyngeal swab eluates were also tested on the Axxin T8-ISO. The developed assay showed sensitivity of 92•31% and 91•89% when tested on the CFX96 and T8, respectively, and specificity of 91•43% and 97•89%, respectively. Interpretation RT-LAMP could be used for SARS-CoV-2 testing and overcomes the challenges of adapting an assay to a point-of-care instrument. Further, the reduced instrumentation cost and complexity, along with the simple workflow, highlight the potential for implementation of RT-LAMP for SARS-CoV-2 testing in resource-limited settings.

There is a need to develop inexpensive, portable and easy-to-use devices for viral sample processing for resource-limited settings. Here we offer a solution to efficient virus capture by incorporating macroporous materials with regular structures into microfluidic devices for affinity chromatography. Two-dimensional simulations were first conducted to investigate the effects of two structures, a nanopost array and a spherical pore network, on nanoparticle capture. Then, the two structures were created in polymers by templating anodic aluminum oxide films and 3D close-packed silica particles, respectively. When the microdevices containing functionalized porous materials were tested for human immunodeficiency virus (HIV) isolation, capture efficiencies of 80–99% were achieved under a continuous flow. Comparatively, functionalized flatbed microchannels captured around 10% of HIV particles. As the characteristic dimensions of the nanostructures are tunable, such devices can be adapted for the capture of different submicron bioparticles. The high capture efficiency and easy-to-operate nature suit the need of resource-limited settings and may find applications for point-of-care diagnostics.