Molecular isothermal techniques for combating infectious diseases: towards low-cost point-of-care diagnostics

Abstract

Nucleic acid amplification techniques such as PCR have facilitated rapid and accurate diagnosis in central laboratories over the past years. PCR-based amplifications require high-precision instruments to perform thermal cycling reactions. Such equipment is bulky, expensive and complex to operate. Progressive advances in isothermal amplification chemistries, microfluidics and detectors miniaturisation are paving the way for the introduction and use of compact ‘sample in-results out’ diagnostic devices. However, this paradigm shift towards decentralised testing poses diverse technological, economic and organizational challenges both in industrialized and developing countries. This review describes the landscape of molecular isothermal diagnostic techniques for infectious diseases, their characteristics, current state of development, and available products, with a focus on new directions towards point-of-care applications.

Keywords::

• ASSURED
• diagnostic
• isothermal amplification
• molecular test
• point-of care

Financial & competing interests disclosure

The authors acknowledge support from The Seventh framework programme of the European Union (FP7-SME-2013, grant 606488). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• An ideal diagnostic test should meet the ‘ASSURED’ criteria: affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and delivered to those who need it.

• Current PCR-based devices, even those intended for application at the point of care, need to be instrumented for performing thermal cycling amplification steps and have limited portability and affordability.

• Molecular isothermal amplification technologies may facilitate development of electricity-free, non- or minimally instrumented point-of-care devices that could be particularly suitable for use in developing countries.

• Proof-of-concept studies in academic laboratory environments have reported that most isothermal techniques have similar or better sensitivity, specificity and rapidness than PCR assays.

• Further studies in real field conditions are needed to validate the promising diagnostic characteristics of isothermal techniques.

• Loop-mediated isothermal amplification, smart amplification process & signal mediated amplification of RNA technology, helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification and nicking and extension amplification reaction are isothermal techniques with mid/high tolerance to inhibitory compounds that allow the use of raw samples without any pretreatment step, which may be an interesting feature for PCR-based point-of-care (POC) testing.

• Isothermal techniques such as loop-mediated isothermal amplification, nicking and extension amplification reaction and recombinase polymerase amplification could be adequate for integration into POC tests due to their rapidness.

• The introduction and use of isothermal POC applications will need to meet the diverse organizational, economic and regulatory challenges before successful adoption.

• Central laboratories in industrialized countries and primary health centers in resource-limited countries are the most plausible scenarios for the deployment of isothermal POC tests in a 5-year view.

Notes