Detection of Falciparum Malaria Imported From Africa With a Highly Speci�c and Sensitive Loop-Mediated Isothermal Ampli�cation (LAMP) Assay

Background: Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as polymerase chain reaction (PCR) are highly sensitive but remain too complex to be used in the ﬁ eld. To complement existing diagnostic methods, a new LAMP assay for the detection and identi�cation of Plasmodium falciparum imported from Africa was developed. Methods: A LAMP assay was developed to amplify the Actin I gene of P. falciparum. Microscopy, Nested PCR and the LAMP assay were conducted on 466 samples of suspected malaria patients imported from Africa to evaluate the new assay’s sensitivity and speci�city. A plasmid construct, cultured P. falciparum, and the clinical samples were used to evaluate the the limit of detection (LOD). Results: Compared to Nested PCR, the sensitivity and speci�city of the nove developed LAMP were 100 % (95% CI 98.54 - 100%) and 99.07 % (95% CI 96.68 - 99.89%) respectively. The LAMP assay was found to be highly sensitive, with the detection limit as low as 10 2 copies/μL and 10-fold higher detection limit than single PCR when performed on serial dilutions of the plasmid construct. The LAMP assay detected 0.01 parasites/μL, when cultured P. falciparum was used as template. The novel LAMP assay detected 1-7 parasites /μL blood in clinical samples(cid:0)which is more sensitively than the commercial product (Loopamp MALARIA Pan/Pf detection kit; Eiken Chemical Co., Tokyo, Japan) for clinical samples detaction of P. falciparum, which detected >20 parasites /μL blood sample. Conclusion:


Background
Malaria is a major cause of morbidity and mortality, leading up to 228 million cases and 405,000 deaths in 2018 (1).With increasing international trade, malaria infection among Chinese has grown.In 2017, 2,858 of 2,861 malaria cases (99.9%) were imported from abroad, of which 822 cases (63.7%) were caused by Plasmodium falciparum (2), which is responsible for most malarial cases in Africa (3).Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi are responsible for other cases.Because different species may require distinct treatment approaches, rapid and accurate diagnosis of P. falciparum is not only crucial for treatment, but also important for disease control, especially for elimination of malaria.
Microscopic examination of blood smears is the most widely used diagnostic approach in the eld and remains the gold standard.This low-cost method allows parasitaemia to be evaluated and permits differentiation among Plasmodium species.Nevertheless, the limit of detection of microscopy is in the range of 10-100 parasites/μL, depending on the health professional's skill and the quality of the smear (4).Rapid diagnostic tests (RDT) (5,6) provide a convenient diagnostic method because no special equipment or special skills are required, and the results can be easily obtained within a short time.
However, microscopy and RDTs cannot reliably detect lower-density parasitaemia (<100 parasites/μL) (7).Molecular diagnostic tools employing DNA ampli cation have the advantage of high accuracy and high sensitivity; of these, nested PCR is considered sensitive and speci c tool for malaria diagnosis(8).Nevertheless, this method is costly and requires trained personnel for its proper implementation.
Loop-mediated isothermal ampli cation (LAMP), originally developed by Notomi et al, is a very sensitive, easy and time saving method (9).The advantages of the LAMP method include high ampli cation e ciency under isothermal conditions (60-65℃), visual judgment based on the turbidity or uorescence of the reaction mixture, and no need for expensive equipment (10).The LAMP method has detected as few as 100 copies of DNA template in blood samples (equal roughly to 5 parasites/μL) (11,12).Currently, two commercial kits are available in the market: LoopAmp malaria (Pan/Pf) detection kit (Eiken Chemical Company, Tokyo, Japan) and Illumigene malaria LAMP assay (Meridian Biosciences, Cincinnati, USA).Each detects symptomatic malaria cases with high sensitivity and speci city (13)(14)(15)(16)(17)(18).However, the evaluation of speci city and sensitivity of LAMP in large Chinese population in China has barely been reported (19)(20)(21)(22), perahaps because commercialized LAMP malaria diagnostic kits are not available in China (https://www.human.de/products/molecular-dx/malaria-lamp).
The aim of this study was to develop a novel LAMP assay for screening large numbers of clinical samples for P. falciparum, and to evaluate its speci city, sensitivity and clinical applicability for the imported malaria from Africa with low-density infections.

Methods
Sample collectionand DNA extraction 3D7 clones of P. falciparum and a Strain H clone of P. knowlesi were obtained from the Malaria Research and Reference Reagent Resource Center (MR4).At lest 3 samples of Toxoplasmagondii and Schistosoma japonicum were obtained from the Department of Pathogen Biology and Immunology, Kunming Medical University.
Recruited patients were 22 to 68 years of age who presented with fever or history of fever within the last 48 h, having symptoms suggestive of malaria, and with tavel history of Africa in two weeks.These samples were obtained at the Shanglin County People's Hospital Guangxi China between 2016 and 2018.Malaria infections were diagnosed by microscopic examination of Giemsa-stained thick and thin blood lms.Data of all cases were recorded, including gender, ethnicity, occupation, microscopic examination results and travel history.The human subject protocol for this study was approved by the Shanglin Hospital Institutional Review Board.Written informed consents were obtained from all included participants.5mL venous blood was collected from every patient and 200uL was used for DNA extraction.DNA was extracted from the studied parasites and blood samples were obtained by using the High Pure PCR Template Preparation Kit (Roche) following the manufacturer's instructions.

Microscopic diagnosis
Thick and thin blood smears were made and stained by Giemsa for observation.For thick smear, Plasmodium parasites were counted in elds totaling 200 WBCs (or 500 WBCs when the initial number of parasites was less than 99) on thick blood lms.Parasite densities were recorded as the average value of parasites per 200 leukocytes as counted by 2 hospital laboratory staff (41).Parasite densities (parasite/ μL whole blood) were estimated by following the World Health Organization (WHO) recommended method (number of parasites /WBC) × 8000 WBC count/μL.(42).

Nested PCR assay
The species of malaria in a sample were determined by using a nested PCR assay.This assay targets the Plasmodium 18s rRNA gene, using previously published primers (43,44).Brie y, for the rst round, a 1µL DNA sample was used as template.For the second round, 1μL of PCR products of the rst round was used as a template.

LAMP primers disgened and and conditions of the LAMP reactions
The full length P. falciparum Actin I gene (GenBank accession no.XM_001350811) is 1,131 bp; a 238 bp fragment (position 4 to 241) was selected as the target sequence for primer design and LAMP detection.Primer design was performed using LAMP Designer 1.15 (PREMIER Biosoft).The primers are listed in Table 1 (patent applying no: 201910611312.9China).Brie y, a reaction contained 1μL of DNA template, 40 pmol each of the FIP and BIP, 5 pmol each of the F3 and B3 primers, 20 pmol each of the LoopF and LoopB, 1μL of uorescent detection reagent (Eiken), 1μL of Bst enzyme (Eiken), 12.5μL RM (Eiken) and DDW was added to make a 25 μL reaction mix.In a thermostatic metal heating block, the mixture was incubated at 63°C for 45 min, and then the reaction was terminated by heating the mixture at 80°C for 5 min.After the reaction, the ampli ed products in the reaction tube were detected under visible light with the naked eye according to the manufacturer's instructions, it changed from clear and orange to green and turbid; or under UV light the solution emited uorescent if it is the positive.The assay results were observed independently by two experimenters who didn't know the PCR/microscopy results.When their evaluation con icted, a third determination was solicited.This experiment was conducted in the laboratory of pathogen biology at Kunming Medical University.
Construction of positive control plasmid DNA PCR products obtained following ampli cation of 3D7 DNA with LAMP primers F3 and B3 (Table 1) were inserted in to pMD TM 19 -T Vector (Takara).The PCR was performed at 94°C for 5 min for denaturation, followed by 35 cycles; 94°C for 30 s, 54°C for 30 s, 72°C for 1 min, and nal extension 72°C for 5 min.PCR products were cloned into vector following its manufacturer's instructions.The recombinant pMD TM 19 -T plasmid DNA cloned P. falciparum Actin I gene fragment was extracted by a Miniprep kit (TIANGEN) from Escherichia coli (DH5a strain), the concentration was measured by a spectrophotometer and copy numbers were calculated by following previously reported method (34).

Speci city and sensitivity of LAMP
The speci city of the assay was tested using genomic DNAs (gDNAs) of various parasites (T.gondii and S. japonicum, P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi).
For sensitivity testing, three DNA templates were prepared.10-fold serial dilutions of constructed plasmid DNA using as the template, the LAMP reaction was compared with the single-PCR assay using F3 and B3 primers (described above), cultivated parasites prepared as previously described (45) and 10-fold serial dilutions of gDNAs of clinical malaria patients using as the templates, the comparison was performed between the LAMP and nested PCR.For comparsion, patented LAMP primers (10, 46) (target gene 18srRNA) were also employed.Each sample was assayed at least 3 times.

Clinical applicability
The clinical applicability of the LAMP was evaluated by using whole-blood samples collected from Shanglin County People's Hospital.Nested PCR was used as the reference as lab standard of sensitivity, speci city.Positive predictive value and Negative predictive value were calculated.Each sample was assayed at least 3 times.

Statistical analysis
All data were analyzed with GraphPad Prism 6.0 and MEDCALP statistical software available at https://www.medcalc.org/calc/diagnostic_test.php.Sensitivity, speci city, and positive and negative predictive values of LAMP were determined using nested PCR as the gold standard for diagnosis of malaria.The concordance response rate (percentage of responses with both positive and both negative results) and Kappa value (k) was determined to measure degree of agreements between two diagnostic test results.Wilcoxon matched-pairs signed rank test and Chi-square test was performed.P value below 0.05 was considered statistically signi cant.LOD calculated by following the report (47).

Evaluation of the speci city of LAMP primers
The speci city of the LAMP primers was investigated by using various parasites, including Plasmodium gDNAs as templates for LAMP.As shown in Figure 1, a green uorescence was detected in P. falciparum gDNA (tube 1) but not when the template DNA derived from other parasite species.

Evaluation of the sensitivity of LAMP primers
To examine the sensitivity of LAMP, three detection methods were used.First, the new LAMP assay was compared to the single-PCR assay using F3 and B3 primers by amplifying 10-fold serial dilutions of plasmid DNA.As shown in Figure 2, ampli cation by real-time LAMP was obtained in reaction tubes containing from 10 9 to 10 2 copies/μL of the DNA template in a 45-min reaction with SYBR Green, with the limit of detection (LOD) was 10 2 copies/μL.In contrast, the LOD for PCR using the F3 and B3 primers was 10 3 copies/μL.Therefore, the sensitivity of the LAMP was 10-fold greater than that of the single-PCR assay.
Comparison of the sensitivity and speci city among the LAMP, nested PCR, and microscopy for malaria diagnosis in the clinical samples A total of 466 suspected malaria clinical samples were used to evaluate the performance of this set of primers for the detection of P. falciparum in clinical samples.Microscopy identi ed parasites with the range in parasite densities of the clinical samples 114-125240 parasite/μL blood.The foreign travel history of these individuals, and the malaria species these identi ed in by nested PCR, are listed in supplemental Table 1.Among the 466 samples, microscopy identi ed P. falciparum in 202, whereas nested PCR and the LAMP assay identi ed the parasite in 251 and 253, respectively (Table 2).Prevalence estimates derived from the two molecular ampli cation methods did not differ signi cantly from each other, but each was signi cantly greater than microscopy (nested PCR vs. Microscopy P = 0.0013; LAMP vs. microscopy P = 0.0008).The comparison of sensitivity and speci city, of microscopy, nested PCR and LAMP is shown in Table 3a, 3b and 3c: all 251 nested PCR-positive samples for P. falciparum were also detected by the novel LAMP (sensitivity of 100%; 95% CI: 98.54 -100%), Kappa = 0.99, 188 were detected by microscopy (sensitivity of 74.90%; 95% CI: 69.06 -80.14%),Kappa = 0.66.213 nested PCR-negative samples for P. falciparum were also negative by the LAMP assay (speci city of 99.07%; 95% CI: 96.68 -99.89%), by microscopy 198 were negative (speci city of 92.09%; 95% CI: 87.64 -95.33%).LAMP successfully detected P. falciparum in the 40 mixed infections.

Discussion
The risk of acquiring locally-transmitted malaria has been virtually eliminated in China, but imported malaria has grown.The diagnosis of malaria at primary clinics has traditionally been performed by microscopic examination of blood smears because of its ease and rapid application.Loop-mediated isothermal ampli cation (LAMP), a novel method developed by Notomi et al. is able to amplify DNA with great e cacy within an hour under isothermal conditions (9); it has been widely used as a diagnostic tool for several parasitic diseases (21,48).Nevertheless, the LAMP method has not been implemented in a large Chinese clinical study (19)(20)(21)(22).In this study, we have done so, and evaluated its speci city, sensitivity and clinical applicability for the detection of P. falciparum imported from Africa for the rst time.
Validating practical assays for detecting imported malaria diagnosis is important for China to reach the goal ofmalaria elimination.We successfully targeted the Actin I gene of P. falciparum for species identi cation on a large sample of clinical isolates and found it to demonstrate high sensitivity and speci city.The P. falciparum Actin I gene was chosen as the target region, because it is a highlyconserved housekeeping gene and differs among the 5 species of Plasmodium.
Compared with the patent primers (Japan), tageting of 18sRNA, our assay demonstrated a comparable LOD (P=0.3466); its sensitivity and speci city were 95% and 99%, respectively (45,49).Our new assay appear more sensitive than those reported previously (19,30).Our LAMP primers showed sensitivity of 1-7 parasites /μL blood from clinical samples, a better performance in clinical patients than reported for the commercial kit, Eiken Loopamp™ MALARIA Pan/Pf detection kit (Eiken Chemical Co., Tokyo, Japan), reported LOD for that assay is >20 parasites /μL blood for P. falciparum (30).The LOD of our LAMP was 100 copies, which was similar with Eiken Loopamp™ (10).Thus the present assay and Eiken Loopamp™ perform comparably when amplifying recombinant plasmid DNA.
Ponce et al. reported that the LOD of Malaria diagnostic kit (Illumigene ® ) was 0.1 Parasites/μL (44).They used serial dilutions of DNA extracted from cultured clones of the 3D7 and W2 strains of P. falciparum.We used the same dilution method with cultured parasites in the initial stage, and we also found it capable of detecting as few as 0.01 parasite/μL.Diluting cultured parasites to evaluate the sensitivity of the reaction does not, however, mirror actual conditions in clinical samples, because culture medium is likely to contain large amounts of DNA from parasites that have died.The concentration of DNA extracted, consequently, may be much higher than the concentration derived from low-parasitemia infections.Furthermore, red blood cell suspensions do not offer the same quantitative accuracy as do diluted DNA solutions.This set of primers could reach the LOD of 10 2 copy/μL when tested with recombinant plasmid DNA, and higher than PCR with F3 and B3.Similar results were obtained by other teams (50).We belive that the recombinant plasmid DNA serves as a more precise means to de ne the sensitivity of such reactions.In our experiment, we used both the clinical samples and recombinant plasmid DNA as template to be used to evaluate the sensitivity; each showed similar results, high sensitivety and speci city.We strongly recommend use the two template types to aid inter-laboratory comparisons and validate clinical relevancy.
The LAMP assay in our study proved to be highly speci c, and there was no nonspeci c reaction to some common parasites or to other Plasmodium species.This set of primers presented 100% sensitivity (95% CI 98.54 -100%) and 99.07 % speci city (95% CI 96.68 -99.89%) compared to nested PCR (P = 0.3466).This is as good or better than previously reported and commercially licensed assays (12,50).Microscopy proved less reliable in cases of low parasitaemia.LAMP and PCR were discordant in two cases, wherein only the LAMP assay was positive.These two cases were con rmed by effective clinical antimalarial treatment, allowing us to conclude that these were true positives.Although this LAMP assay was carried out in the laboratory, it can be used in the eld due to its simple equipment requirements and uncomplicated operating procedures.Compared with nested PCR, LAMP reaction time was only 45 minutes, which greatly improved the detection e ciency and provided timely evidence for clinical medication strategy.
Based on the resultsobtained, this LAMP assay offers an alternative for molecular diagnosis for P. falciparum infection in non-malaria-endemic regions, like China.This will strengthen the control and prevention of imported malaria in China.

Table 2 .
The positive outcome 466 clinical malaria cases diagnosed by three methods