A pilot evaluation of alternative procedures to simplify LAMP-based malaria diagnosis in field conditions

Highly-sensitive and field-friendly diagnostic tools are needed for accurate detection of low-density malaria infections. Although loop-mediated isothermal amplification (LAMP) fulfills these conditions, operational challenges are still encountered during pilot population screenings in remote settings when employing Loopamp™ MALARIA Pan/Pf detection kit (Eiken Chemical Co.). This study evaluates different procedures for the simplification of sample preparation and result reading steps of current LAMP protocols. The reference 'Boil & Spin' (B&S) pre-amplification procedure was compared to three alternative methods, along with a colorimetric staining protocol based on malachite green. Results suggested that the B&S supernatant transference step may be omitted without an impact on test performance, even when colorimetry was incorporated to facilitate results visualization. Procedures skipping centrifugation and/or heat-incubation were proved to be compatible with LAMP-based malaria DNA detection, but resulted in a low-to-moderate decrease in sensitivity and ambiguous result interpretation for the most straightforward protocol. Nevertheless, all simplified LAMP methods could still reach lower limits of detection than the currently used tools for malaria mass-screening (i.e. microscopy and rapid tests), indicating that these alternative strategies may deserve further consideration. This evaluation, therefore, demonstrates the feasibility of skipping some of the main procedural bottlenecks of LAMP-malaria protocols, a much-needed achievement to make point-of-care implementation of molecular diagnostics a reality.