Though the global distribution and disease burden of P. vivax
is considerable, current molecular diagnostic methods that accurately detect P. vivax
are limited. The RealAmp platform is a potential tool for using molecular diagnostic techniques in the field. The assay is simple, and provides rapid results through real-time data collection. This combination is ideal for developing countries that may not have the capacity and infrastructure to maintain PCR-based assays at every health care facility. The objective of this study was to design and test the utility of a novel set of LAMP P. vivax
-specific primers using different target DNA sequences. The previously described genome sequence data-mining methods 
, adapted in this study, have proved to be useful in designing novel specific primers for P. vivax
detection. Recent studies have developed new P. falciparum
diagnostic LAMP primers based on mitochondrial DNA 
and the apical membrane antigen-1 (AMA-1) gene sequence 
was used to amplify P. knowlesi
DNA. These new targets will help optimize the RealAmp assay for accurate detection of all malaria parasite species.
We have noted previously that Plasmodium
subtelomeric regions harbor repetitive regions that are well suited to species-specific diagnostic targets 
. As the majority of Pvr64 copies are not assembled onto chromosomes, and the short assemblies containing these copies do not contain telltale signs of subtelomeric regions such as vir
genes, we cannot be certain whether or not Pvr64 is a subtelomeric repeat. However, one chromosomal copy does occur ~ 500 kb interior to the end of the assembly, arguably outside of the nebulously defined “subtelomeric” region. Though Pvr64 has some overlapping regions in other Plasmodium
parasites such as P. falciparum
and P. knowlesi
, the primers developed in this study did not show any cross reactivity with other examined malaria parasites. While Pvr64 is similar to the 18S rRNA target in that it is relatively low-copy and not species-specific, it offers a superior, more sensitive RealAmp target than the 18S rRNA gene family, perhaps due to its comparatively high degree of conservation within the P. vivax
The r64 primer set has shown reasonable sensitivity and specificity in detecting P. vivax among the clinical isolates used, accurately detecting 70 out of the 74 positive samples. It is possible that the four P. vivax samples that this primer set missed had low parasite density. Unfortunately, we were not able to determine this since we did not have any parasitemia data for the samples used in this study. Since P. vivax is genetically similar to several simian malaria parasites, we also tested the r64 primer set on eight simian parasites and observed no cross-reactivity. These primers were also able to amplify 13 P. vivax strains from different geographic locations. It is therefore likely that this assay will be able to detect P. vivax strains in malaria-endemic regions around the world.
Attempts to use published P. vivax
LAMP primers by Han et al
. in the RealAmp platform failed to yield consistent results, and the sensitivity was not as high as previously reported 
. Possible explanations for this observation is the fact that different nested-PCR methods were used as the gold standard in calculating the sensitivity and specificity and that different detection methods were used in these studies. We also tested another published set of P. vivax
LAMP primers by Tao et al
. However, these primers cross reacted with the other human-infecting Plasmodium
species (P. falciparum, P. ovale
and P. malariae
), hence were not considered for further evaluation.
In some studies, SYBR Green was shown to have inhibitory effects on DNA amplification when used in high concentrations 
. We did not observe any inhibitory effect with SYBR Green in our assays. However, we obtained better amplification plots with SYTO-9 with lower background noise and in some cases, a better distinction from baseline fluorescence. This suggests that SYTO-9 is a better dye than SYBR Green for RealAmp assays and is possibly due to perfect spectral match with the detector. This is an important component of the assay as it will reduce misclassification of cases and non-cases by the users in the field.
One of the limitations of this primer set is its low sensitivity. Our data suggests that the limit of detection of the r64 primer set is around 125 parasites/µL when a quantified standard was used. This level of sensitivity might be sufficient for case management of malaria but not for the detection of sub-clinical malaria cases which is important during the pre-elimination phase of malaria. Efforts are underway to improve these limits of detection to enable detection of lower parasitemia. However, compared to nested PCR as a gold standard, this method performed reasonably well with approximately 95% sensitivity in detecting the clinical samples in a single assay.
As previously described, the RealAmp assay 
has several advantages that make it a good choice for field-based molecular diagnosis of malaria. However, some hurdles need to be overcome for the RealAmp assay, and any molecular assay, to be truly field-usable. For example, a major caveat of molecular assays is the sample preparation phase; many assays require that the template DNA is purified and isolated. While the use of boiled whole blood was shown to give decent results with the RealAmp assay 
, additional studies are required to further simplify this step. In addition, the cold chain requirement for reagents used by many molecular assays needs to be addressed. Studies are underway to investigate if the RealAmp reagents can be combined into a “kit” which would further simplify the protocol, reduce the number of steps for the end user, and minimize chances of user-introduced errors in performing this assay. Lyophilized reagents that can be reconstituted at a later time would reduce preparation time and address the issue of reagent storage without a cold chain supply.
In conclusion, this study demonstrates that the P. vivax
LAMP primers developed here have the ability to accurately detect P. vivax
infections on the RealAmp platform. Furthermore, the previously described genome sequence data-mining methods have proved to be useful in designing primers not just for conventional PCR 
but also for LAMP assays. This technique offers great potential in identifying novel primer sets for detection of other Plasmodium