Nucleic acid amplification-based diagnostics

Molecular detection of malaria

• qualitative or quantitative parasite detection,
• determination of the multiplicity of infection,
• genotyping to distinguish recrudescence from reinfection, and
• detection of drug resistance mutations.

Currently, WHO recommends that the use of NAATs be considered only for research purposes until more evidence is available on the public health impact of detecting and treating low density infections.

Types of nucleic acid amplification tests

Polymerase chain reaction (PCR) – including nested (n), quantitative (q) or real-time reverse transcription (RT-PCR), loop mediated isothermal amplification (LAMP), and quantitative nucleic acid sequence-based amplification (QT-NASBA) – are among the key NAATs that have been developed to detect malaria. NAATs can offer quantitative as well as qualitative output, and many tests have also been developed to enable specific detection of both sexual- and asexual-stage parasites. The 18S ribosomal RNA gene has unique sequences that enable the identification of all 5 malaria species infecting humans – Plasmodium falciparum, P. vivax, P. ovale, P. malaraie and P. knowlesi – and is therefore commonly targeted for amplification.

NAATs may differ according to required equipment and infrastructure, pre-amplification sample processing, reaction temperature, time to result, ease of use, limit of detection, or cost. However, all tests involve broadly the same steps for their utilization. Most require a nucleic acid extraction step followed by amplification, which may occur at a single temperature or by cycling through different temperatures. Depending on the method selected, detection or quantification may be determined in any of the following ways:

• visually on an agarose gel illuminated under ultraviolet light, 
• through examination of reaction tubes in a turbidimeter, or 
• through incorporation of fluorescent labels in the reaction, allowing continuous real time monitoring of product production during amplification, followed by comparison with known titrated controls. 

Quality assurance of NAATs

While NAAT offer sensitive detection, reproducibility has been reported to be imperfect, particularly when detecting low density parasitaemias. To strengthen reliability and comparability of data, an international external quality assurance programme has been established with the aim of improving standards of performance of NAATs for malaria. The service provides external quality assurance reference material from erythrocyte stage infection at a range of parasite densities, as lyophilized and dried blood spots. The first round of testing began early 2017. For more information on participation, please write to MalNAATEQA@who.int.

More information on NAATs