Malaria diagnosis using a combined system of a simple and fast extraction method with a lyophilised Dual-LAMP assay in a non-endemic setting

ABSTRACT

Malaria is a parasitic disease distributed in tropical areas but with a high number of imported cases in non-endemic countries. The most specific and sensitive malaria diagnostic methods are PCR and LAMP. However, both require specific equipment, extraction procedures and a cold chain. This study aims to solve some limitations of LAMP method with the optimization and validation of six LAMP assays, genus and species-specific, using an easy and fast extraction method, the incorporation of a reaction control assay, two ways (Dual) of result reading and reagent lyophilization. The Dual-LAMP assays were validated against the Nested-Multiplex Malaria PCR. A conventional column and saline extraction methods, and the use of lyophilized reaction tubes were also assessed. A new reaction control Dual-LAMP-RC assay was designed. Dual-LAMP-Pspp assay showed no cross-reactivity with other parasites, repeatability and reproducibility of 100%, a significant correlation between parasite concentration and time to amplification and a LoD of 1.22 parasites/µl and 5.82 parasites/µl using column and saline extraction methods, respectively. Sensitivity and specificity of the six Dual-LAMP assays reach values of 100% or close to this, being lower for the Dual-LAMP-Pm. The Dual-LAMP-RC assay worked as expected. Lyophilized Dual-LAMP results were concordant with the reference method. Dual-LAMP malaria assays with the addition of a new reaction control LAMP assay and the use of a fast and easy saline extraction method, provided low limit of detection, no cross-reactivity, and good sensitivity and specificity. Furthermore, the reagent lyophilization and the dual result reading allow their use in most settings.

KEYWORDS:

• Malaria
• Plasmodium
• Molecular diagnosis
• LAMP
• Real time LAMP

Acknowledgements

The authors are grateful to all personnel from the Malaria & Emerging Parasitic Diseases Laboratory and to the staff of the Department of Parasitology from the National Microbiology Center (ISCIII, Spain), for their support and valuable comments on this manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ contribution

JMR, AMR, SRH and APA conceptualized the study. JMR and AMR designed the experiments; JMR provided training and supervision for the procedures; AMR, LBA, CCR and MLS performed the experiments. All authors contributed to the writing of the final version of the manuscript. All authors read and approved the final manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/20477724.2023.2232595

Additional information

Funding

This work was supported by the Spanish Strategic Health Action (AESI-ISCIII) [Grant number PI17CIII/00035, PI22CIII/00033]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Alexandra Martín Ramírez was supported by a grant from ISCIII-Río Hortega [Grant number CM17CIII/00012].