Point-Of-Care or Point-Of-Need Diagnostic Tests: Time to Change Outbreak Investigation and Pathogen Detection

Abstract

In the recent years, the progress of international trade and travel has led to an increased risk of emerging infections. Around 75 percent of the pathogens causing these infections are of animal origin. Point-of-care tests (POCT) and point-of-need tests (PONT) have been established in order to directly provide accurate and rapid diagnostics at field level, the patient bed-side or at the site of outbreaks. These assays can help physicians and decision makers to take the right action without delay. Typically, POCT and PONT rely on genomic identification of pathogens or track their immunological fingerprint. Recently, protocols for metagenomic diagnostics in the field have been developed. In this review, we give an overview of the latest developments in portable diagnostic methods. In addition, four mobile platforms for the implementation of these techniques at point-of-care and point-of-need are described. These approaches can provide reliable diagnostics and surveillance, especially in low resource settings as well as at the level of one health.

Keywords: diagnostics; field applicable diagnostics; isothermal amplification; lateral flow assay; mobile laboratory; point-of-care; point-of-need.

Figure 1

Structure and type of a lateral flow immunoassay. (A) Schematic design: 1, sample pad; 2, conjugate pad; 3, membrane; 4, test line; 5, control line; 6, adsorbent pad. (B) Indirect lateral flow immunoassay. (C) Competitive lateral flow immunoassay. (D) Sandwich lateral flow immunoassay. The principle of a lateral flow immunoassay (LFIA) is as follows—the sample is brought onto the sample pad and flows in the opposite direction by adsorption. While the sample passes the conjugate pad, a labelled target-specific antibody binds to the target molecule in the sample (antigen or antibody). Afterwards, the labelled antibody–target complex is immobilized to the membrane by a specific capture molecule (antibody or antigen) adhered to the membrane at the test line. The unbound labelled antibodies are captured at a control line by immobilized antibodies. In the case of a positive sample, the accumulation of the labelled antibodies leads to a coloration of both test lines. In the case of a negative sample, only the control line is colored. The adsorbent pad takes up the excess liquid. In the case of pathogens or targets, which are not suitable for the indirect or sandwich designed LFIAs, a competitive layout can be applied. This results in only one colored test line in case of a positive test.

Figure 2

The loop-mediated isothermal amplification (LAMP) is based on a so called auto-cycling strand-displacement DNA synthesis using a strand-displacing DNA polymerase and two pairs of target-specific primers. The inner primers contain two sequences each corresponding to the sense and anti-sense (rc) strand of the target DNA, respectively, linked with a TTTT spacer. The inner primers initiate a complementary synthesis of the target DNA while the outer primers (FP1 and RP3) initiate a strand-displacement synthesis resulting in the release of single-stranded DNA linked by inner primers. The single-stranded DNA forms stem-loops by self-annealing of the corresponding sequences and acts as template for exponential amplification [23].

Figure 3

The recombinase polymerase amplification (A) employs a recombinase enzyme, a single-stranded binding protein, a strand-displacing (ss-DNA) polymerase, and a pair of target-specific primers. The primers form together with the recombinase enzyme the recombinase—primer complex which allows them to invade the double-stranded helix of the target DNA at the respective recognition site. The strand-displacing polymerase elongates the primers while the ss-DNA binding protein stabilizes the displaced strand in order to prevent self-annealing and ejection of the primers by branch migration. Real-time detection of amplification (B) is achieved by a sequence specific probe consisting out of a tetrahydrofuran abasic–site mimic (THF) flanked by fluorophore and quencher-labelled nucleotides as well as an extension blocker at the 3´ end. As the probe pairs with the complimentary sequence, a double-strand specific exonuclease slices the THF and the fluorophore is dissociated resulting in a signal [24]. Detection of RPA amplicon is in a lateral flow format (C,D). The use of a 5′end Biotin-labelled reverse primer in the RPA reaction leads to an antigenic-tagged reverse strand. When the 5-Carboxyfluorescein (FAM)-labelled probe binds to the complimentary strand a double-strand specific endonuclease slices the THF. The extension blocker is released and the remaining oligonucleotide can act as primer resulting in a FAM- and biotin-labelled amplicon (C), which can be detected in a lateral flow sandwich format (D, [25]).

Figure 4

(A) MinION sequencing device (Oxford Nanopore technology, Cambridge, UK) and (B) the principle of nanopore sequencing. A protein pore is embedded in a membrane set under a certain electrical current. As the DNA molecule passes through the pore each nucleotide is recognized by the individual change caused in the voltage.