Proof of Concept for a Portable Platform for Molecular Diagnosis of Tropical Diseases

Abstract

or adtcosta@ ibmp.org.br.Although molecular diagnostics is well established in clinical laboratories, its full potential has not been extended to field settings. Typically, diagnostic real-time quantitative PCR (qPCR) reagents require temperature-controlled transportation and storage. Furthermore, thermocyclers are bulky and fragile, requiring good infrastructure for optimal operation. These major hurdles strongly limit use of molecularbased tests in low-resource scenarios. Herein, Trypanosoma cruzi or Plasmodium spp. DNA were detected with qPCR using commercial equipment (ABI7500 instrument) and a prototype platform comprising a portable device and a silicon chip, named Q3-Plus. In addition, a ready-to-use reaction format, where all qPCR reagents are stored on plate or on chip, was compared with the traditional freezer-stored format. No significant differences were observed in detecting T. cruzi or Plasmodium spp. DNA between thermocyclers, as well as between reagents' formats, for storage periods of up to 28 days (at 2 C to 8 C or 21 C to 23 C, respectively). When challenged with patients' samples, the Q3-Plus system performed as efficiently as the standard equipment for Plasmodium spp. DNA detection, showing it to be a valuable solution to malaria point-of-care diagnostics. Detection of T. cruzi DNA in chronic patients' samples using the Q3-Plus system yielded approximately 50% efficiency relative to the ABI7500. These results are essential to support future endeavors to bring molecular diagnostics to the point of care, where most needed.

Demonstration of a fast and easy sample-to-answer protocol for tuberculosis screening in point-of-care settings: A proof of concept study

“…The low number of samples analyzed in the present study limits our ability to draw definitive conclusions, but the results obtained with the optimized protocol showed good agreement with results obtained by reference diagnosis methods such as culture or other molecular tests, providing the basis for future studies. The ready-to-use reactions performed similarly to the conventional ones, exhibiting similar lower detection limits, as previously shown for detection of T. cruzi or Plasmodium spp DNA [12]. After evaluation of larger cohorts, we suggest that the ready-to-use reactions could be implemented in places with unreliable power supply, as in urban suburbs and rural areas of high TB burden countries of Brazil, India, Indonesia, China, Philippines and Pakistan.…”

“…An overview of the full procedure is outlined in Fig 3, showing a direct comparison to the general workflow of commercially available spin-column extraction kits. Although the ASSURED requirements have been met to a large extent in platforms such as GeneXpert Ultra or BioFire, the Q3-Plus system shows some advantages over both the aforementioned instruments and the standard benchtop ABI7500 in terms of equipment and reaction costs, reaction volume, user friendly interface, fast temperature ramping, and shorter total reaction time [12,20]. Some of these features will be further discussed below.…”

“…all qPCR reagents (enzymes, buffers, oligonucleotides, dNTPs, etc.) and 5 μL of in substitution for molecular-grade water in [12,[17][18][19]. The gelification solution contains three classes of components, each with specific functions within the mixture and the process: (i) trehalose and melezitose (400 mg/mL each), to protect the biomolecules from the desiccation process by stabilizing and reducing the activity of water in the solution; (ii) lysine (0.75 mg/mL), a free radical scavenger which inhibits oxidizing reactions between carbonyl or carboxyl groups and the biomolecule's amino or phosphate groups; and (iii) glycogen (200 mg/mL), an inert polymer which creates a broader protective matrix.…”

“…The gelification solution contains three classes of components, each with specific functions within the mixture and the process: (i) trehalose and melezitose (400 mg/mL each), to protect the biomolecules from the desiccation process by stabilizing and reducing the activity of water in the solution; (ii) lysine (0.75 mg/mL), a free radical scavenger which inhibits oxidizing reactions between carbonyl or carboxyl groups and the biomolecule's amino or phosphate groups; and (iii) glycogen (200 mg/mL), an inert polymer which creates a broader protective matrix. These components define a sol-gel mixture that prevent the loss of the tertiary or quaternary structure during the desiccation process, thus helping maintaining the biomolecules' activity upon rehydration [12,17]. The final concentration of each component of the gelification mixture used in this study was trehalose 160 mg/mL, melezitose 80 mg/mL, glycogen 40 mg/mL and lysine 0.15 mg/mL.…”

“…A rapid, simple, efficient, less expensive and ideally equipment-free sample preparation would not only bring down the cost of molecular assays but also mitigate the challenge of access to low-resource settings [9,10]. Furthermore, the reagents used in molecular assays require special storage and transportation, which further increase their costs [11,12]. Largely, the successful integration of sample preparation with ready-to-use reagents and portable qPCR systems would solve the main hurdles in point of care testing [9,10,13].…”