The Development of a 3D Printer-Inspired, Microgravity-Compatible Sample Preparation Device for Future Use Inside the International Space Station.

Biological testing on the International Space Station (ISS) is necessary in order to monitor the microbial burden and identify risks to crew health. With support from a NASA Phase I Small Business Innovative Research contract, we have developed a compact prototype of a microgravity-compatible, automated versatile sample preparation platform (VSPP). The VSPP was built by modifying entry-level 3D printers that cost USD 200-USD 800. In addition, 3D printing was also used to prototype microgravity-compatible reagent wells and cartridges. The VSPP's primary function would enable NASA to rapidly identify microorganisms that could affect crew safety. It has the potential to process samples from various sample matrices (swab, potable water, blood, urine, etc.), thus yielding high-quality nucleic acids for downstream molecular detection and identification in a closed-cartridge system. When fully developed and validated in microgravity environments, this highly automated system will allow labor-intensive and time-consuming processes to be carried out via a turnkey, closed system using prefilled cartridges and magnetic particle-based chemistries. This manuscript demonstrates that the VSPP can extract high-quality nucleic acids from urine (Zika viral RNA) and whole blood (human RNase P gene) in a ground-level laboratory setting using nucleic acid-binding magnetic particles. The viral RNA detection data showed that the VSPP can process contrived urine samples at clinically relevant levels (as low as 50 PFU/extraction). The extraction of human DNA from eight replicate samples showed that the DNA extraction yield is highly consistent (there was a standard deviation of 0.4 threshold cycle when the extracted and purified DNA was tested via real-time polymerase chain reaction). Additionally, the VSPP underwent 2.1 s drop tower microgravity tests to determine if its components are compatible for use in microgravity. Our findings will aid future research in adapting extraction well geometry for 1 g and low g working environments operated by the VSPP. Future microgravity testing of the VSPP in the parabolic flights and in the ISS is planned.

Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease.

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

PrintrLab incubator: A portable and low-cost CO2 incubator based on an open-source 3D printer architecture.

Growth in open-source hardware designs combined with the decreasing cost of high-quality 3D printers have supported a resurgence of in-house custom lab equipment development. Herein, we describe a low-cost (< $400), open-source CO2 incubator. The system is comprised of a Raspberry Pi computer connected to a 3D printer controller board that has controls for a CO2 sensor, solenoid valve, heater, and thermistors. CO2 is supplied through the sublimation of dry ice stored inside a thermos to create a sustained 5% CO2 supply. The unit is controlled via G-Code commands sent by the Raspberry Pi to the controller board. In addition, we built a custom software application for remote control and used the open-source Grafana dashboard for remote monitoring. Our data show that we can maintain consistent CO2 and temperature levels for over three days without manual interruption. The results from our culture plates and real-time PCR indicate that our incubator performed equally well when compared to a much more expensive commercial CO2 incubator. We have also demonstrated that the antibiotic susceptibility assay can be performed in this low-cost CO2 incubator. Our work also indicates that the system can be connected to incubator chambers of various chamber volumes.

A Rapid SARS-CoV-2 RT-PCR Assay for Low Resource Settings.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. However, it generally requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers. As a pandemic, COVID-19 has spread indiscriminately, and many low resource settings and developing countries do not have the means for fast and accurate COVID-19 detection to control the outbreak. Additionally, long assay times, in part caused by slow sample preparation steps, have created a large backlog when testing patient samples suspected of COVID-19. With many PCR-based molecular assays including an extraction step, this can take a significant amount of time and labor, especially if the extraction is performed manually. Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests, and the backlog we currently experience can be reduced drastically. Furthermore, our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in locations where sophisticated laboratory instruments are not available. Our USD 300 set up achieved rapid RT-PCR using thin-walled PCR tubes and a water bath setup using sous vide immersion heaters, a Raspberry Pi computer, and a single servo motor that can process up to 96 samples at a time. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. These findings can help rapid COVID-19 testing to become more accessible and attainable across the globe.