Pakistan-US Science and Technology Cooperation Program Phase 7 (2017 Deadline) Health security: Point of care, multiplexed molecular detection of infectious diseases endemic in Pakistan US Partner: Haim Bau, University of Pennsylvania Pakistan Partner: Muhammad Shabir, International Islamic University   Project summary This project will train Pakistani researchers in the use and implementation of a newly developed platform for the detection of hepatitis C, malaria, and HIV in blood samples, pathogens endemic in Pakistan and associated with significant morbidity, mortality, and socio-economic cost. The platform will also be expandable for use with other targets and samples, including environmental and agricultural samples and food products. Progress Reports 2020: This collaborative program between the International Islamic University (Islamabad, Pakistan) and the University of Pennsylvania (Philadelphia, PA, USA) has developed point of need diagnostic systems to detect the presence of nucleic acids associated with pathogens or diseases in biological and/or environmental samples. Our diagnostic system is comprised of a disposable cassette and a processor. To enable rapid prototyping, the Penn team used 3D-printing to fabricate our cassettes while the Pakistani team used layered manufacturing. Our cassette accepts a sample mixed with lysis and binding buffer; isolates, immobilizes, and concentrates nucleic acids; purifies the captured nucleic acids, and carries out loop mediated isothermal enzymatic amplification (LAMP) to increase the number of target molecules and enable their detection. Our disposable cassette is comprised of a slider and a housing. The slider hosts a silica membrane for the isolation and immobilization of nucleic acids, a reaction chamber for the amplification of nucleic acids, and a passive actuator to compress blisters (located in the housing). The housing accommodates blisters with stored liquids; needles to pierce the blisters; absorption pads to provide capillary imbibition and waste storage; a mechanism to seal the reaction chamber prior to nucleic acid amplification; and microfluidic conduits. In operation, the user introduces a sample into the cassette. The sample filters through a nucleic acid capture membrane. This membrane is propelled through blister-actuated wash stations to purify the nucleic acids and remove inhibitors. Then, the nucleic acids are eluted with water into the reaction chamber, blended with a reaction mix, and the reaction chamber is sealed. The cassette mates with a processor that provides thermal control to incubate the LAMP process at about 65oC and imaging to monitor the amplification process via fluorescent dye in real time. The cassette can be operated either manually or it can be actuated with an electric motor. During the project, the USA team hosted a faculty and a doctoral student from Pakistan at UPenn for short term visits. 2019: Our team has developed a two-stage isothermal enzymatic amplification assay for the detection of nucleic acids associated with the human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The analytical performance of this assay was verified at Penn and patient samples were tested in Pakistan (single-plex) for HIV and HCV on benchtop equipment. Patients’ test results obtained with the Penn isothermal assay were critically compared with standard quantitative PCR tests (the gold standard). A 3D-printed slider cassette was designed, fabricated, and tested. The current cassette accommodates a single reaction chamber equipped with a nucleic acid isolation membrane at its inlet. When lysed samples filter through the isolation membranes, nucleic acids bind to the membranes while the rest of the sample is discharged to waste. Our isolation membrane allows one to decouple sample volume from reaction chamber volume, facilitating higher sensitivity than common in rapid molecular tests. We incubate the chip with a simple custom-made processor that maintains the chip at a desired temperature for the duration of the test. The slider cassette consists of slider and a cassette body. The slider houses the nucleic acid isolation membrane and the reaction chamber. We have developed a blister system to store and discharge wash solutions through the nucleic isolation membrane to remove any substances that may interfere with nucleic acid amplification. The sample preparation was demonstrated to be compatible with the requirements of nucleic acid amplification. Additionally, we have designed and fabricated a thermal control system that provides the incubation temperature for nucleic acid amplification. 2018: Our team has developed a two stage isothermal enzymatic amplification assay for the detection of nucleic acids associated with the human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The analytical performance of this assay was tested at Penn and patient samples were tested in Pakistan (single-plex) for HIV and HCV on benchtop equipment. Patients’ test results obtained with the Penn isothermal assay were critically compared with standard quantitative PCR tests (the gold standard). A 3D-printed microfluidic chip was designed, fabricated, and tested. The chip accommodates multiple reaction chambers, each for a single-plex assay. Each reaction chamber is equipped with a nucleic acid isolation membrane at its inlet. When lysed samples filter through the isolation membranes, nucleic acids bind to the membranes while the rest of the sample is discharged to waste. Our isolation membrane allows one to decouple sample volume from reaction chamber volume, allowing higher sensitivity than common in rapid molecular tests. We incubate the chip with a simple custom-made processor that maintains the chip at a desired temperature for the duration of the test. Nucleic acid amplification is monitored by detecting emission intensity from either intercalating fluorescent dye or luciferous dye that emits light without excitation with a USB-based microscope or the ubiquitously available smartphone camera. The operation of the chip requires manual pipetting of wash solutions. To minimize manual operations, the team is developing a slider cassette that will house all wash solutions and wash. The slider cassette consists of slider housing the nucleic acid isolation membrane and reaction chamber and a house hosting blisters with wash solutions and absorption pads. We have demonstrated that the slider cassette successfully incubates enzymatic amplification. The development of the cassette has not been completed, however, and will continue in the next grant period.