In-house validation of a lamp kit for diagnosis of Plasmodium, Plasmodium falciparum and Plasmodium vivax in Vietnam

Up to day, Malaria is still considered as a public health problem in tropical and subtropical regions. In 2018, there were approximately 228 million estimated cases of malaria identifi ed globally [1]. According to a report of the national malaria prevention program, in 2019 Vietnam had 4,665 malaria parasites detected by cyanoscopy reduced 3.08% compared to 2018. Rate of parasite / 1,000 people was 0.048. The number of malaria cases gradually decreased through 10-year period from 2010 to 2019. In 2019 malaria patients decreased by 89.16% compared to 2010. Malaria parasites concentrated mainly in the Central and Central Highland provinces. The number of malaria parasites may be lower than they actually are, due to the presence of asymptomatic malaria and the low density of parasites below the detection threshold of a microscope; According to some unpublished reports in Vietnam, this rate can reach 30-50% [2]. Currently, there are three main groups of methods applied in the diagnosis of malaria parasites: Giemsa stained blood smear, rapid diagnostic test (based on immune method), Molecular biology techniques like PCR, Real time-PCR [3]. Giemsa-stained smear test is still the Abstract

gold standard in the confi rmed diagnosis of malaria case [4].
This method is widely used, most popular in many countries around the world, with an average detection threshold of about 50 -100 parasites/μL of blood from all Plasmodium spp, low test costs, performed even in laboratory conditions and fi eld testing [1]. However, this method is diffi cult to detect cases with low parasite density, the testing staff must trained, must be retrained regularly, especially in areas with low prevalence of malaria or eliminate areas where there is little opportunity for testing [5][6][7]. Rapid diagnostic tests (RDTs) are also widely used, especially in areas where microscopy sites have not yet been available, but RDTs still have limitations such as low parasite detection threshold, false positive rate, and long positive time [5,7,8]. The common disadvantage of these two methods is the low threshold for detection of malaria parasites [9,10]. Therefore, it is estimated that a large number of people detection or rapid test (RDTs) [4]. In particular, traditional PCR techniques such as Nested-PCR, Seminested Multiplex-PCR based on the target gene of 18S -rRNA are widely applied in the diagnostic studies of malaria for high sensitivity and specifi city [11][12][13]. The detection was determined to be between 1-5 parasites /μl of blood [13]. The Real time-PCR technique has a high sensitivity at the LOD > 0.5 KST /μl of blood [14]. However, current DNA amplifi cation techniques are timeconsuming, complex and not fi eld-friendly.
To eliminate malaria globally, strategic measures are needed to increase the ability to detect sources of disease. The technical limitations of current testing methods, which requires the development of a highly sensitive diagnostic kit which has a detection threshold comparable to that of DNA amplifi cation techniques in the laboratory, but requires no expensive equipment, faster testing times, is easy for training and can be done in the fi eld. The LAMP technique is a molecular biology technique that uses heat to amplify single strands of DNA, fi rst described by Notomi, et al. [15]. The method has a high amplifi cation effi ciency in 15-60 minutes [15]. And then Han ET, et al. [16], developed LAMP to detection of four Plasmodium species for clinical diagnosis [16]. Imai Kazuo, et al. (2017) reported the novel diagnostic method for malaria using LAMP and MinION™ nanopore sequencer [17]. Jaymin C, et al. (2013) developed the RealAmp technique, a LAMP technique combined with real-time isotherms to quickly detect malaria parasites. This study also built a primer pair to detect P. vivax by RealAmp method [18]. Compared with other molecular methods such as PCR, real time PCR ..., LAMP has the advantage of having similar accuracy, simple implementation, especially short execution time and can detect results by naked eye [11, [19][20][21][22]. Therefore, LAMP is often used to create quick diagnostic kits which are widely popular in the world. LAMP has been utilized to detect malaria parasites mainly using the conventional 18S ribosomal RNA (18S rRNA) gene as the target sequence [6,12,16,[23][24][25][26][27]. In this study, we have developed and evaluated the LAMP technique for the detection of Plasmodium genus, Plasmodium falciparum and Plasmodium vivax from whole blood and dry blood spot.

Samples
All of samples used for positive control purposes were Whole Blood (WB) and Dry Blood Spot (DBS) from human infected with Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. The samples were collected from human Gia Lai province, Vietnam. There were confi rmed from WHO EQA program. There used DNA from Plasmodium as positive control and DNA from other parasites was also extracted to determine the specifi city.
The use of the human and the collection of samples were approved by the bioethics committee of National Institute of Malariology, Parasitology and Entomology (NIMPE).

DNA extraction protocol
Using the QIAamp DNA microkit of Qiagen (Germany) extracted total DNA of Plasmodium species from WB and DBS samples. We followed the extraction procedure is performed according to the manufacturer's instructions.

LAMP primer design
Primers used for the LAMP assay were designed based on a highly conserved region of Plasmodium genus, Plasmodium falciparum, Plasmodium vivax genome. Genbank sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi) ( Table 1)   With P. falciparum and P. vivax no cross reactivity was observed with any of the tested simian malaria parasite species or with the malaria-negative human DNA control ( Figure 1) Table 2.     To determine the lower detection limit, 10-fold serial dilutions of each plasmid DNA were amplifi ed. The detection limit for LAMP was 2,97 x 10 -2 copies for P. falciparum and 2,85 copies for P. vivax, respectively. The amplifi cation products were visualised on an agarose gel as a ladder of multiple bands.
Although microscopic examination by Giemsa microscopy has been identifi ed as the gold standard for the diagnosis of malaria parasites. But the detection threshold of this technique is from 20 to 100 parasites/1μl of blood; much higher than the detection threshold of LAMP technique which is about 2-3 parasites/μl of blood; If we compare these two techniques directly, the sensitivity will be very low such as Table 3.
We therefore compared the results of the LAMP test with the qPCR technique, which has similar sensitivity and specifi city is to be recognized as a country eliminating malaria by 2030 [28]. To achieve this goal, the National Malaria Control       Program of Vietnam has been implementing many appropriate strategies such as strengthening monitoring, detection, diagnosis and timely treatment. Every year in Viet Nam, the health system conducts tests of more than 2.2 million blood smears, nearly 500,000 rapid diagnostic tests for malaria [28].
The disadvantage of these two techniques is the low detection threshold. Therefore, it is estimated that a large number of people with low-density parasites are not detected. In areas where seasonal malaria is transmitted, people infected with low-density parasites below the detection threshold of blood smear and RDTs are both a regular source of transmission, but also a source of parasite storage through a low infectious season to a high infectious season [28]. Interventions with investigation and treatment to reducing malaria transmission have limited to rely on the detection of antigens using RDTs to identify asymptomatic cases of malaria patients. good specifi city and a lower detection limit of 25 parasites/μL [13,[31][32][33][34]. The detection limit of our LAMP methods for all Plasmodium species, P. falciparum and P. vivax was 2.89 × 10 -1 , 2.97 × 10 -2 and 2.85 copies/reaction, respectively (5μL of sample DNA could be loaded per reaction). Therefore, the theoretical detection limit of the LAMP assay was equivalent to that of the previously described Loopamp™ MALARIA Pan/Pf and P.v Detection Kit.
The sensitivity and specifi city of LAMP assay were evaluated with 200 clinical samples and used qPCR as a gold standard.
The results show that the sensitivity and specifi city of all assay are over 97%, proving that the LAMP assay in this study has high sensitivity and specifi city such as PCR and qPCR. However, PCR and real-time PCR methods require expensive equipments such as thermal cyclers or real-time PCR systems for accurate temperature control, expensive cost and diffi cult to apply in the fi eld.

Conclusion
LAMP is a molecular method with high sensitivity and specifi city. LAMP products are visible to the naked eye after the addition of the MG color indicator, which allows LAMP to be easily deployed in the fi eld in medical facilities.