Evaluation of Visual LAMP detection of Plasmodium falciparum and non- Plasmodium falciparum CURRENT

Background: detect and effect. Method; According to the 18s rRNA sequence of the malaria, the LAMP primer of Plasmodium falciparum and non-Plasmodium falciparum were designed using Primer Explorer V5 software. The method of Visual LAMP detecting malaria was formation. The sensitivity, specificity and amplification efficiency were tested, compared with the Nest-PCR. Result: The filter papers of blood from 47 patients with Plasmodium falciparum and 49 with non-Plasmodium falciparum were detected using Visual LAMP. The patients with Plasmodium falciparum were all positive. In the patients with non-Plasmodium falciparum , false negative rate was 2.1%. In the 10 patients with Leishmaniasis, 8 with Schistosomiasis japonicum, 38 healthy human, none was positive. The results using visual LAMP were consistent with Nest-PCR (Kappa value = 0.956). All of the Tt in Visual LAMP are less than 60 min, especially the Tt of Plasmodium falciparum is 18.2 min. When adding calcium pyridoxine indicator during the Visual LAMP detection, the progress of the reaction to different substes of malaria delayed of about 9-23 min. Conclusion: LAMP is efficient and visible, deserved the prospect of application in the on-site and primary medical institutions.


Background
Malaria is one of the most important parasitic infectious diseases which threaten human life safety [1] .
There are 4 kinds of malaria parasites, of which Plasmodium falciparum is the most harmful, toxic, and with the highest fatality rate [2,3] . According to The World Malaria Report 2018, $3.1 billion was invested in control and eradication of malaria in 2017, with a total of 219 million malaria cases worldwide and 435,000 deaths. In Africa, South-East Asia, the Eastern Mediterranean, the Western Pacific and China, Plasmodium falciparum accounted for 99.7%, 62.8%, 69%, 71.9% and 63.7% in all malaria cases, respectively [4,5] . Malaria control in China is at the elimination phase. Importation of malaria remains widespread due to increased travel and population movements to malaria-endemic areas. To reduce malaria complications, fatality and transmission, it is important to diagnose malaria 3 correctly [1] . The common detection methods of malaria parasites are mainly optical microscope examination and RDT. In the chronic infection stage, due to the malaria parasite emias always maintain low density, optical microscope examination and RDT leak detection [3,6] . The RDT is insensitive to Plasmodium vivax and Plasmodium ovale. The patients with asymptomatic Plasmodium falciparum infection may be malaria-infected reservoir [7,8] . Therefore, a high sensitivity diagnostic methods able to detect low-density malaria parasite emias for the elimination of malaria is critical [6,9] .There is urgent need for extremely sensitive and field-based diagnostic system [10] .
Molecular diagnosis is a highly sensitive method for detecting malaria infection [7,9,11] . For example, PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, but it requires expensive equipment [7] . The sensitivity of the ring-mediated so-mediated temperature amplification (LAMP) method is close to or exceeds the NEST-PCR [6,7,12,13]

2.4
Healthy people: blood from the healthy volunteers were collected, filter paper blood spots were made, naturally dried, saved at 4℃.
3. DNA extraction: filter paper with dried blood were droplets by 3mmx3mm, according with The OMEGA D3096 micro-DNA extraction kit operation, extract DNA of malaria (50 μl), saved at -20℃.

Discussion
Malaria is an infectious disease caused by different types of Plasmodium, which live in red blood cells (RBC) and destroy host erythrocyte, leading to fever and/or anemia, or even kidney failure, brain coma and death. Accurate diagnosis of malaria infection is critical. Microscope examination of blood smears is still considered the gold standard for the diagnosis of malaria, which was labor-intensive, time-consuming [7,10,16] , with detection limit of 100 malaria parasites/µl [10,16] . It was usually unable to detect low-density and mixed infections, the sensitivity and specificity of which depends on the skill of the technician, quality and staining quality of blood smear, and so on [3,17] . Compared with molecular diagnostic methods, in malaria endemic areas, only half of malaria patients were correctly diagnosed. The WHO external assessment of the malaria parasite's mirror test capacity in China showed that the correct rates of negative, Plasmodium falciparum (P.f),, Plasmodium vivax (P.v) were 92.5%, 78.3%, 70.8% respectively [20] . RDT is facilitate and fast, with detection limit of 100 plasmids/ μl [19,21] . It shows high sensitivity to plasmid infections of more than 100 plasmids/μl(median 94.3%), sensitivity of which decrease significantly with a density of less than 100 plasmids/μl (median 74.1%) [21] . Meantime it shows limited sensitivity when detecting low-density infection [16,22] , and less sensitivity to non-P.f P.v as 66.0-88.0%, P.o as 5.5-86.7%, P.m as 21.4-45.2% [19] . PCR is one of the most sensitive and reliable methods for detecting DNA of malaria, with a high sensitivity and specificity limit to 1-10 malaria parasites/μl [10,11] . However, it requires technical expertise and infrastructure which limit its wide range of applications. LAMP has been used in clinical diagnosis of a variety of infectious diseases, including malaria. It is considered to be faster, more operational and cost-effective than PCR. At the same time, it has been reported that in testing malaria, LAMP own a similar sensitivity and specificity to PCR [7,10,11,23,,24] . It is capable of identifying low-density malaria infections by LAMP, which cannot be detected by the microscope [7] . Sensitivity of LAMP is several orders of magnitude higher than by microscope and RDT [21] .
At present, detecting malaria by LAMP is mainly to detect the genus malaria and Plasmodium falciparum, the primer is depend on 18s rRNA region of the malaria parasite species. In this study, 3 As worldwide countries set targets of malaria elimination, accurate diagnosis, optimal treatment, stop of transmission, and reducing mortality of malaria are becoming critical [25,26] . As a result of the sudden decline in local patients and the gradual elimination of malaria, there are a trend of degradation in the clinical diagnosis and screening capacity of malaria. The prevalence of infrequent diagnosis, with some cases ranging from onset to the determination of diagnosis for more than 30 days. At the same time, due to increasing travel and population movements to malaria-endemic areas, imported malaria is still widespread [21] . Particularly, asymptomatic malaria infection and lowdensity malaria parasite emias are hard to be detected by microscope examination and RDT due to below the detection threshold [11,24] , causing misdiagnosis or delayed diagnosis [3,21] . But they could maintaining transmission of malaria as infected intermediate host [24,26] . It has been reported that subclinical malaria accounts for 70-80% of all malaria infections [11] . Therefore, it is a challenge to detect the asymptomatic malaria parasite infection and low density malaria parasite emias in malaria prevention and control [8,24] . Stopping transmission of malaria requires detecting methods with high sensitive. The LAMP shows high sensitivity and specificity, by which asymptomatic and asymptomatic malaria infections could be diagnosed accurately, and low-density infections that cannot be detected by conventional diagnosis methods could be identified [26] . At the same time, LAMP is fast, easy to operate and effective which provides the possibility for clinical diagnosis and monitoring of malaria.

Conclusion
Visual LAMP is highly sensitive and specific to the diagnosis of malaria,and the results using visual LAMP are consistent with Nest-PCR.During the malaria elimination stage,visual LAMP provides a portable, sensitive and specific method for on-site inspection.

Declarations
Ethics approval and consent to participate This study was certified by the Ethics Committee of the Wuhan Center for Disease Control and Prevention. The informed consent of all patients and healthy people was obtained.

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.  Effect of the indicator on the LAMP of P.v Effect of the indicator on the LAMP of P.m