Table 1.

Operational features and performance of molecular methods.

Diagnostic method	Operational features	Performance	Advantage	Disadvantage	Throughput	Optimal setting for field use
Nested PCR	•Two sets of primers used in successive reactions; therefore, more expense, time and potential contamination than single-step PCR	•Limit of detection: at least 6 parasites/μL for blood spots •More sensitive than single-step PCR for the four main Plasmodium species •Hands-on time to result: 3 h; total time: 10 h	•Simple, it reduces the degree of non-specific binding, •The specificity of the PCR reaction is enhanced by reducing the non-specific binding with the help of the two sets of primers	•Time consuming, •Needs more reagents such as extra set of primers, high chance of contamination	High	Field applicable
Multiplex PCR	•Simultaneous, multiplex PCR to detect the presence of multiple Plasmodium species	•Limit of detection: 0.2–5 parasites/μL •Hands-on time to result: 2 h; total time: 4.5 h	•More information with less sample, cost effective, time saving, high accuracy, less pipetting errors, less contamination.	•Low amplification efficiency, complex, variability in efficiency in different templates and poor universality	High	Field applicable
Quantitative PCR	•Rapid amplification, simultaneous detection and quantification of target DNA by use of specific fluorophore probes	•Limit of detection: 0.02 parasites/μL for genus-level identification, 1.22 parasites/μL for P. falciparum detection •Hands-on time to result: 1 h; total time: 2.5 h	•Fast, efficient, and gives a qualitative result	•It is not cost effective and complex due to simultaneous thermal cycling and fluorescence detection.	High	Field applicable
Nucleic acid sequence-based amplification	•Assay includes a reverse transcriptase step, less inhibition than PCR. Isothermal method. Can be used to quantify gametocytes. Detects all four Plasmodium species, targeting 18S rRNA. Result by fluorescence	•Limit of detection: 0.01–0.1 para- sites/μL per 50-μl sample •Result within 90 min (not including extraction time of about an additional 90 min)	•A major advantage of NASBA is the production of single stranded RNA amplicons that can be used directly in another round of amplification. •It supports the detection of human mRNA sequences without the risk of DNA contamination •It helps in better RT-PCR reaction as it offers faster amplification kinetics.	•Expensive thermocycling equipment is not needed as the reaction occurs isothermally at 41 °C.	High	Field applicable
CLIP-PCR	•Highly sensitive method rRNA of the plasmodium parasite can be released from the blood and then captured onto 96-well plates. Finally, quantified through the number of ligated probes which bounds to it.	•Capture and ligation probe PCR(CLIP-PCR) which enables to detect the parasite density in blood as low as 0.01 parasites per microliter of blood.	•CLIP-PCR is highly sensitive, and can detect malaria concentrations as low as 0.01 parasitized cells/microliter of blood	•Expensive, and complex	High	Field applicable
LAMP	•Boil-and-spin extraction can be used, with amplification by isothermal method. Result determined by turbidity or fluorescence. Sensitivity increases by including mitochondrial targets. Genus-level targets, P. falciparum and P. vivax.	•Limit of detection: 0.2–2 parasites/μL •Results within 30 min with a tube scanner	•LAMP is cost-effective and requires minimal capital equipment investment	•Restricted availability of reagents and instruments, no multiplex capability and limitations related with primer design. •Does not allow the inclusion of an internal PCR inhibition control (IC)	High	Field applicable
HtLAMP	•A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform. The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wave length of 600 nm.	•Limit of detection is 2.5 parasites/μL	•Simple, highly sensitive, and more specific. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98%	•LAMP based assays are in general expensive	High	Field applicable