There is a growing request for faster and more sensitive diagnostic methods for malaria. Conventional methods, such as microscopy and RDTs, lack sensitivity, while PCR-based methods, although very sensitive , require highly trained personnel and expensive equipment. In contrast, LAMP-based methods have proven to have a similar or higher sensitivity than PCR methods but with the need for less training and resources [16, 21]. Alethia® Malaria kit is a qualitative commercial assay able to detect Plasmodium spp. based on LAMP technology [22, 23]. Furthermore, the kit includes the components to isolate DNA from whole blood by centrifuge-free methods . In our study, the comparison between LAMP and NM-PCR assays, with just two discrepant samples (1.9%), showed a very good correlation corroborated with the high values of sensitivity, specificity and predictive values, as well as the kappa value (Table 2). Regarding the two discrepant results, it is very difficult to assess the accuracy of the LAMP method because any meaningful evaluation must be involved in comparison with other methods of diagnosis, in this occasion the NM-PCR, which might themselves be wrong. The false-positive result could be due to DNA contamination during sample processing [24, 25] or even to be a true-positive . In each extraction set, a free-DNA sample was included and it did not produce amplification by the LAMP method, so possibly the second option was more feasible. The false-negative result in the sample infected with P. malariae may be due to a low parasitaemia of the original sample or that the LAMP assay exhibits deficiencies for the detection of this species. In this study, only four P. malariae-infected samples were analyzed, of which only two (50%) were correctly characterized, meanwhile another was being considered negative and the last gave an invalid result. Expanding the number of samples infected with P. malariae would be necessary to find the correct answer.
Similar good results in specificity and sensitivity have been shown in more studies in non-endemic countries [2, 14, 15, 17]. In a study performed in France , they obtained 100% of sensitivity and 98.13% specificity using real-time PCR as the reference method. Studies performed in North America obtained excellent sensitivity compared to microscopy  and PCR . Moreover, the Alethia® Malaria kit has also been evaluated in Senegal obtaining high sensitivity (97.2%) and a good specificity . However, neither of these studies tested any P. knowlesi specimens. In our study, the nine P. knowlesi-infected samples were detected, without any invalid or false-negative results, confirming the ability of the Alethia® Malaria LAMP to detect the main five Plasmodium human species.
Nine samples out of 114 provided invalid results. Several authors have described a similar situation where it was not possible to obtain a valid result with this LAMP assay [2, 16]. According to the manufacturer’s instructions, some possible reasons for the invalid results may be inhibitory specimens, improper sample preparation, reagent failure, instrument failure, dirty device or improperly seated. In our study, samples with an invalid result were obtained at the beginning of the study but not at the end, suggesting that the technologist’s level of training played a factor in the results probably due to improper mixing of the blood samples and buffers.
The Alethia® Malaria kit showed a very good LoD value for P. falciparum, 0.075 parasites/µl, similar to reported for several malaria Nested PCRs [18, 26]. Other reports obtained different LoD depending, possibly, on the origin of the samples used; 2 parasites/µl  and 0.5 parasites/µl  with conditions similar to ours, or 0.1 parasites/µl using serial dilutions of P. falciparum cultures .
Several malaria LAMP assays have been described previously [8–11], most of them “homemade” assays with reproducibility problems and with difficult-to-pass quality assessment controls. Conversely, commercial LAMP kits for malaria detection present the reagents in lyophilized form to enhance stability under ambient conditions, facilitating the use and decreasing the need for high training. Furthermore, the Alethia® Malaria kit incorporates the components to purify DNA from whole blood and an internal reaction control to discriminate false negatives from inhibition reactions. Unfortunately, contrary to the best feature of LAMP technology, the reaction must be run on a specific device. Despite this, given our results, with excellent sensitivity and specificity and with a reduced time of diagnosis, Alethia® Malaria assay could be used as a good screening diagnosis method for malaria, as other authors have pointed out previously [12, 16, 17].