Asymptomatic Malaria Infections Epidemiological Survey At Endemic Sites Of Myanmar

Objective: The world health organization's malaria reports in the last three years show a slight increase in the number of cases of malaria worldwide(216 million in2016,219 million in 2017,228 million in 2018),and more than 400,000 deaths from malaria infections each year.Southeast Asia accounted for 7 percent of malaria cases and 6 percent of deaths globally in 2016.In Southeast Asia,the six countries within the Greater Mekong Subregion(GMS)(China,Myanmar,Thailand,Laos,Vietnam,Cambodia)have a regional goal of malaria elimination by 2030.To achieve this goal,control efforts to continuously shrink the malarious areas have intensified,whereas in areas where malaria transmission had been interrupted,increased surveillance has been implemented to maintain the malaria-free status and prevent malaria reintroduction.The Myanmar malaria burden still remains high within the GMS,and malaria transmission persists in most international border regions.This poses a serious threat to the neighboring countries and greatly hinders regional malaria elimination.Typically,most malaria infections in endemic areas are asymptomatic,while confirmed cases of acute malaria are just “the tip of an iceberg”.Although asymptomatic malaria infections rarely cause acute illness,they can infect mosquitoes,making them potential hosts for continued malaria transmission.Because asymptomatic infections and complex transmission patterns can lead to the persistence of malaria,drug-resistant strains prevalent in asymptomatic infections may be the basis for the rapid spread of resistance.In Myanmar,where multiple firstline treatments are used to treat Plasmodium falciparum infections,all first-line drugs experience a delayed clearance of weakened insecticidal effects,and the emergence and spread of drug-resistant strains threatens the local goal of eliminating malaria.Antimalarial resistance of Plasmodium falciparum tends to occur in areas with low infection rates,particularly in southeast Asia and South America,and then spreads to areas with high infection rates in sub-saharan Africa.Currently,data on resistance to asymptomatic infections are limited.Reliable estimates of asymptomatic malaria epidemics and molecular epidemiological studies of resistance to these parasite hosts and timely monitoring of the spread of antimalarial resistance are critical tomaintaining the latest advances in malaria control and achieving the regional goal of eliminating malaria.This study collected filter paper blood samples from people scattered in several towns in different areas of Myanmar to analyze the infection rates of asymptomatic Plasmodium parasites and mutations in antimalarial resistance genes in Plasmodium falciparum infections.These results will provide updated information on asymptomatic Plasmodium infection in multiple areas of Myanmar and the status of drug resistance in Plasmodium falciparum malaria,which is important for developing strategies to eliminate Plasmodium falciparum infection.Methods: Part 1: In this study,cross-sectional surveys were conducted during the rainy season of 2017 in four townships(Bilin,Thabeikkyin,Banmauk and Paletwa)of Myanmar with divergent annual malaria incidences.A total of 1991 volunteers were recruited from local villages and Plasmodium subclinical infections were estimated by light microscopy(LM),rapid diagnostic tests(RDTs)and nPCR.The nPCR analysis was performed with a modified pooling strategy that was optimized based on an initial estimate the infection prevalence.Data were analyzed by SPSS v.22.0.Malaria prevalence was calculated by crosstabs.The odds ratio(OR)was calculated for binary variables and Chi-square or Fisher's exact tests were used where appropriate.P< 0.05 was considered statistically significant.The sensitivity and specificity of uRDT and cRDT in detecting slide-positive and nPCR-positive P.falciparum infections in our study population were calculated by crosstabs with LM or nPCR as the gold standard.Part 2: Three cross-sectional studies were conducted in Myanmar at Laiza(Kachin State),Banmauk(Sagaing Region)and Paletwa(Chin State)townships in 2015,2017-2018 and 2017,respectively.genetic variations in drug resistance markers of Plasmodium falciparum were determined in parasites from asymptomatic populations located in three geographically dispersed townships of Myanmar by PCR and sequencing.Mutations in dihydrofolate reductase(pfdhfr),dihydropteroate synthase(pfdhps),chloroquine resistance transporter(pfcrt),multidrug resistance 1(pfmdr1),multidrug resistance protein 1(pfmrp1),and Kelch protein 13(k13).We evaluated the quality of sequences by examining the chromatograms.The sequences were aligned using ClustalW in MEGA7.0.26.The samples with monoclonal P.falciparuminfections were used for single nucleotide polymorphism(SNP)and haplotype analysis.The SNP data from different genes were analyzed using Microsoft Soft Excel 2007 and SPSS 22.0.Pearson's Chi-square test or Fisher's exact test were used to determine statistical significance(P< 0.05).The haplotype network was constructed by DnaSP 6 and Network software 5.01.0 using the median joining algorithm.Results: Part 1: The overall malaria infection prevalence based on all methods was13.9%(277/1991)and it differed drastically among the townships,with Paletwa in the western border having the highest infection rate(22.9%)and Thabeikkyin in central Myanmar having the lowest(3.9%).As expected,nPCR was the most sensitive and identified 226(11.4%)individuals with parasite infections.Among the parasite species,Plasmodium vivax was the most prevalent in all locations,while Plasmodium falciparum also accounted for 32% of infections in the western township Paletwa.Two RDTs based on the detection of the hrp2 antigen detected a total of 103 P.falciparum infections,and the ultrasensitive RDT detected 20% more P.falciparum infections than the conventional RDT.In contrast,LM missed the majority of the subclinical infections and only identified 14 Plasmodium infections.Part 2: Mutations in dihydrofolate reductase(pfdhfr),dihydropteroate synthase(pfdhps),chloroquine resistance transporter(pfcrt),multidrug resistance 1(pfmdr1),multidrug resistance protein 1(pfmrp1),and Kelch protein 13(k13)were present in92.5,97.6,84.0,98.7,68.3 and 61.4% of the parasites,respectively.The pfcrt K76 T,pfmdr1 N86 Y,pfmdr1 I185 K,and pfmrp1 I876 V mutations were present in 82.7,2.5,87.5 and 59.8% isolates,respectively.The most prevalent haplotypes for pfdhfr,pfdhps,pfcrt and pfmdr1 were 51I/59R/108N/164 L,436A/437G/540E/581 A,74I/75E/76T/220S/271E/326S/356T/371 I and 86N/130E/184Y/185K/1225 V,respectively.In addition,57 isolates had three different point mutations(K191T,F446 I and P574L)and three types of N-terminal insertions(N,NN,NNN)in the k13 gene.In total,43 distinct haplotypes potentially associated with multidrug resistance were identified.Conclusion: 1.Cross-sectional surveys identified considerable levels of asymptomatic Plasmodium infections in endemic populations of Myanmar with P.vivax becoming the predominant parasite species.The pooling scheme designed for nPCR analysis offers a more practical strategy for large-scale epidemiological studies of parasite prevalence.2.Analysis of molecular markers of drug resistance among asymptomatic P.falciparum infections revealed a high prevalence of mutations linked to drug resistance in Myanmar.High mutation frequency in pfdhfr,pfdhps and pfcrt means serious resistance to SP and CQ in these areas,which supports the ACT being used for P.falciparum treatment in Myanmar.Molecular surveillance of antimalarial resistance might be helpful for developing and updating guidance for the use of antimalarials in Myanmar.