Table 3.

Modern (PCR/LAMP-based) methods used for malaria detection and evidence of use in developed countries.

Modern Methods	Specimen Used	Description	Invasive/Non-Invasive	Point of Care/Molecular/Other	Advantages	Disadvantages	Developed Countries	References
Direct conventional PCR	Blood	With plasmodium cytochrome oxidase III gene (COX-III) as target, direct conventional PCR is conducted on bloodspot samples. Results are visualized on a gel.	Invasive	Molecular	High Sensitivity; faster than nested; does not require DNA isolation	Requires much expertise and expensive	USA	[21]
Nested Polymerase Chain Reaction (PCR)	Blood	Using different primer pairs to run 2 sequential amplification reactions. Plasmodium genomic DNA extracted from dried blood spots	Invasive	Molecular	High sensitivity and specificity	Time consuming, expensive, requires much expertise	Thailand, USA, Brazil, United Kingdom, Austria	[13,16,18,21,22,23,24,25]
Droplet Digital PCR (ddPCR)	Blood, Serum	DNA extracted from blood and serum samples are analyzed using the ddPCR method, which is based on water–oil emulsion droplet technology	Invasive	Molecular	High sensitivity using blood samples	Low sensitivity using serum samples; expensive	Italy, Thailand	[26,27]
Photo- Induced Electron transfer PCR (PET-PCR)	Blood	Total DNA is extracted from dried blood spots and PCR performed using photo-induced electron transfer fluorogenic primers	Invasive	Molecular	High sen-sitivity for parasite identification and characterization.	Requires much expertise and is expensive	USA	[15]
Fluoresen-ce reson-ance energy transfer (FRET) real time PCR	Blood	Real-time PCR utilizing FRET whereby fluorophores are brought in close proximity after hybridization is performed on DNA extracted from lyophilized blood samples targeting the 18S rRNA gene	Invasive	Molecular	High sensit-ivity, and allows for simultaneous quantitative and species-specific detection	This specific protocol could not differentiate between P. vivax and P. knowlesi; expensive	United Kingdom, Austria	[22]
SYBR Green Real-Time PCR-RFLP Assay	Blood	Real-time PCR using sybr green dye that binds to all double-stranded DNA followed by restriction fragment polymorphism to differentiate species	Invasive	Molecular	High sensitivity	Meltcurve required in PCR since Sybr green alone can be non-specific; expensive	Sweden	[28]
Hair qPCR	Head hairs	Hairs without roots are taken from patients and qPCR assay conducted	Non-invasive	molecular	Requires no special trans-port/storage conditions for samples	Sensitivity lower than when blood samples are used	Spain	[29]
Insulated Isothermal PCR (iiPCR)	Blood	PCR is performed in a portable device using an assay based on the Rayleigh–Bénard convection method	Invasive	Molecular/point of care	Portable, easy and fast operation; direct interpretation	Not as sensitive as qPCR	Malaysia	[30]
Lab Chip Real Time PCR (LRP)	Blood	DNA is extracted from collected blood samples and a portable LRP device is used to detect malarial parasites based on lab-on-chip technology	Invasive	Molecular/point of care	High sensitivity and specificity. Fast and cost effective	Risk of false negatives higher than traditional real-time PCR	Korea	[31]
Pv-mt Cox PCR	Blood	DNA is extracted from collected blood samples and qPCR with mitochondrial gene target is carried out	Invasive	Molecular	More sensitive in the detection of P. vivax	Expensive	Brazil	[32]
PvLAP5 and Pvs25qRT-PCR assays	Blood	Extracted RNA is subjected to quantitative reverse transcription PCR	Invasive	Molecular	Suitable assay for the determination of human transmission reservoir	Expensive	Panama	[33]
Other Quantita-tive PCR (qPCR)	Blood	Real-time PCR performed using primers targeting different regions and SYBR green or probe-based technology on DNA extracted from whole blood	Invasive	Molecular	High sensitivity and rapid	Extreme caution needed to prevent contamination; expensive	France, Canada, USA Columbia Germany, Brazil, China, Malaysia	[34,35,36,37,38,39,40,41,42,43,44]
Dry LAMP system (CZC-LAMP)	Blood	Blood samples are analyzed directly without extraction using the assay made up of dried reagents	Invasive	Point of care/molecular	High sensitivity and specificity; no need for prior extraction	Not widely available		[45]
Particle Diffusometry (PD)-LAMP	Blood	PD, which senses images, is combined with LAMP on a smartphone-enabled device to detect low levels of parasitemia	Invasive	Point of care/molecular	Sensitivitities higher than RDTs and similar to qPCR	Sensitivity slightly lower than nested PCR	USA	[46]
LAMP and MinION™ nanopore sequencer	Blood	Primers targeting the 18S–rRNA gene of all five human Plasmodium species are generated and samples subjected to LAMP. Min-ION™ nanopore sequencer is used on amplicons to identify Plasmodium spp.	Invasive	Molecular	Highly sensitive, and simple	Expensive	Japan	[47]
Other Loop-mediated isothermal amplification (LAMP),	Blood	Extracted DNA is subjected to loop-mediated isothermal amplification with a variety of detection methods	Invasive	Point of care/molecular	Simple, low cost; can be used in resource-limited settings and point-of-care settings	Some cannot quantify par-asite density; some are insensitive towards low parasitemia and mixed infections	France, Korea, Thailand Italy, Brazil Spain, Mala-ysia, Japan, Peru, USA	[26,34,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63]