Fully automated point-of-care differential diagnosis of acute febrile illness

Abstract

Background In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk (“FeverDisk” for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device. Methodology/Principal findings A sample volume of 200 μL per FeverDisk was used. In situ extraction with pre-stored reagents was achieved by bind-wash-elute chemistry and magnetic particles. Enzymes for the loop-mediated isothermal amplification (LAMP) were pre-stored in lyopellet form providing stability and independence from the cold chain. The total time to result from sample inlet to read out was 2 h. The proof-of-principle was demonstrated in three small-scale feasibility studies: in Dakar, Senegal and Khartoum, Sudan we tested biobanked samples using 29 and 9 disks, respectively; in Reinfeld, Germany we tested spiked samples and analyzed the limit of detection using three bacteria simultaneously spiked in whole blood using 15 disks. Overall during the three studies, the FeverDisk detected dengue virus (different serotypes), chikungunya virus, Plasmodium falciparum, Salmonella enterica Typhi, Salmonella enterica Paratyphi A and Streptococcus pneumoniae. Conclusions/Significance The FeverDisk proved to be universally applicable as it successfully detected all different types of pathogens as single or co-infections, while it also managed to define the serotype of un-serotyped dengue samples. Thirty-eight FeverDisks at the two African sites provided 59 assay results, out of which 51 (86.4%) were confirmed with reference assay results. The results provide a promising outlook for future implementation of the platform in larger prospective clinical studies for defining its clinical sensitivity and specificity. The technology aims to provide multi-target diagnosis of the origins of fever, which will help fight lethal diseases and the incessant rise of antimicrobial resistance.

ImmunoDisk—A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored

“…Lyophilization or freeze-drying is often used to store proteins (alone, i.e., without beads) due to the good stabilization achieved with this method, even though it is costly and technologically complex [ 61 ]. Concerning beads alone, we have demonstrated protocols for pre-storage in the case of magnetic beads for nucleic acid extraction and purification [ 62 , 63 , 64 ]. However, the BFPD-IA that is integrated within the ImmunoDisk involves protein-coupled magnetic and fluorescent beads.…”

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice

“…In the disks that we tested, there was no case where the S. marinus was not detected, thereby confirming qualitatively that the sample-to-answer process functioned successfully. The simultaneous detection of several bacteria, including the process control, within a single sample is a major achievement of the platform, which until now had demonstrated its capability for infectious diseases that are associated with either one or two pathogens, such as sepsis [ 18 ], tropical infections [ 20 ] and respiratory tract infections [ 19 , 21 ].…”

“…Then, 20 µL of magnetic beads with 10 µL of 250 mg/mL trehalose were pipetted into the corresponding chamber ( Figure 2 , #3), and 3.5 µL of each primer/probe reaction mix with 0.5 µL of 1 M trehalose (final concentration 50 mM) were pipetted into each reaction chamber ( Figure 2 , #7). The Teflon coating and the drying of the magnetic beads and primers/probes followed a previously published protocol (1 h at 50 °C) [ 20 , 41 , 42 ]. The lyopellets containing the amplification reagents were manually inserted into the disk.…”

“…The OralDisk microfluidic cartridge integrates all the biochemical reagents needed for fully automated analysis, namely: (i) customized buffers and microfluidic-optimized magnetic particles for the purification of bacterial DNA; (ii) amplification reagents in a lyophilized form; and (iii) POC-ready real-time qPCR TaqMan primers/probes for specific bacterial detection. The OralDisk is an application-specific version of the centrifugal microfluidic LabDisk platform, which has already demonstrated its utility in applications where a single infectious pathogen is to be detected [ 18 , 19 , 20 , 21 ]. This small-scale technical feasibility study demonstrates for the first time the platform’s implementation in the field of oral health, where multiple bacteria may be present simultaneously in the oral cavity and whole saliva is used as diagnostic specimen.…”

Novel lab-on-a-disk platforms: a powerful tool for molecular fingerprinting of oral and respiratory tract infections

“…Such high degree of universality pays off at the level of scalable manufacturing. The cartridge remains easily adaptable to the detection of diverse targets by exchanging the primers/probes in the geometrically discrete reaction chambers, instead of re-engineering the entire cartridge [16,23].…”