Study On Epidemiological Characteristics Of Malaria Asymptomatic Carriers Along The China-Myanmar Border

Background and study objective:Identification of all malaria infections including asymptomatic carriers for interrupting transmission is one of the core strategies for malaria elimination.Malaria asymptomatic carriers are considered as "reservoir of infection",which has an important role in malaria transmission and infectious sources management,especially in the malaria elimination programs.The border between Yunnan Province and Myanmar is the key challenging area in the National Malaria Elimination Action Plan of China,and the role of the asymptomatic infection in this area was more and more concerned.However,the published research and evidences to address this issue were limited.This study aimed to figure out the prevalence,epidemiological characteristics and risk factors for malaria asymptomatic carrier along the China-Myanmar border,which provided scientific evidence for the implementation of the core strategy of"case tracking,focus clearance" to eliminate malaria and consolidate the achievements of eliminating malaria in this area.Study design:This study included three parts.Firstly,we compared the sensitivity and consistency of different methods for malaria detection,such as microscopy,real time fluorescent PCR and ultrasensitive PCR,and selected the most sensitive method to identify the asymptomatic infections.Then we implemented cross-sectional investigation along the China-Myanmar border to understand the prevalence of asymptomatic malaria infection on both sides of the border(Part ?).A detailed questionnaire including demographic information,occupation,human mosquito exposure,malaria history and other information related to Plasmodium spp.infection were collected and analyzed by univariate and multivariate analysis to elaborate the epidemiological characteristics and influencing factors for asymptomatic infections.The polymorphism of immune molecule gene and molecular markers of drug resistance were detected as well(Part ?).A cohort study of asymptomatic carriers was carried out to investigate the persistence and oscillations of asymptomatic infection,as well as its risk factors(Part ?).Methods:Dried blood spots were collected from the healthy residents in communities along the China-Myanmar border,and were tested by three different methods including light microscopy,real-time fluorescent PCR and ultrasensitive PCR.The positive rate and consistency of these three methods were compared to select the most sensitive one to identify the asymptomatic malaria infection.A cross-sectional investigation was carried out in four villages/internally disabled people(IDP)along the China-Myanmar border.Demographic data,including age,gender,occupation,as well as travel history,mosquito exposure,recent malaria history was collected.The epidemiological characteristics and risk factors of asymptomatic malaria were analyzed by univariate and multivariate analysis.The polymorphism of molecular markers associated with drug resistance in asymptomatic infections was analyzed as well.TLR SNP loci(TLR9 T1486C,TLR9 G1174A,TLR5 R392Stop)were detected by MALDI-TOF MS,and the influence of allele and genotype polymorphism of TLR SNP locus on asymptomatic infections was analyzed.A cohort study of asymptomatic malaria was carried out to understand the persistence and oscillations of asymptomatic carriers,as well as its risk factors were analyzed by Kaplan-Meier plots and Cox proportional risk regression model.Results:Of the three methods,ultrasensitive PCR showed the highest positive rate for identifying the asymptomatic infections.Among 387 samples,38 were detected positive by ultrasensitive PCR with positive rate 9.82%;13 were positive by real time fluorescent PCR with positive rate 3.36%;only 6 were positive by light microscopy with positive rate 1.55%.The consistent rates of ultrasensitive PCR with the real time fluorescent PCR and the light microscopic detection were 93.54%and 91.73%,respectively.The positive cases detected by the fluorescent PCR and the light microscopic also could be detected by the ultrasensitive PCR,which indicated that the ultrasensitive PCR was the most sensitive to detect asymptomatic infections.A cross-sectional study for asymptomatic infections was carried out in four villages/settlements(3 sites in Myanmar and 1 site in China)on both sides of the China-Myanmar border by ultrasensitive PCR.The results showed that the prevalence of asymptomatic infection in the study area was 8,81%,all the tested positive infection was P.vivax.The prevalence of asymptomatic malaria infection(9.60%)in Myanmar sites was significantly higher than that in China(3.40%)(x2=6.13,P=0.01).The prevalence of asymptomatic infection varied depending on the incidence of malaria symptomatic infection in China site.The incidence of malaria in 2014 in China site was 0.40%,the prevalence of asymptomatic malaria was 9.75%,while the incidence of malaria in 2014 was 0%,and the prevalence of asymptomatic infection was 3.40%(?2=5.543,P=0.02).Although malaria incidence in Myanmar sites also declined during the same period,there was no significant difference in prevalence of asymptomatic malaria infection between 2014 and 2019,which was 10.00%and 9.60%,respectively.The prevalence of asymptomatic malaria infection in male(10.91%)was significantly higher than that in female(7.21%)(x2=4.78,P=0.03).There was also a significant difference in the prevalence of asymptomatic malaria at different ages(X2=14.89,P=0.02).The highest rate was found in the age of 51-60 yr(17.57%).The peak of the asymptomatic infection rate in male population was the 41-50 yr age group with 22.58%,whereas the peak of female was the 51-60 yr age group with 15.69%,and the ages group of 11-20 yr with 8.77%.Univariate and multivariate analysis showed that gender,main living/working place,the greater travelling distance every week were the influencing factors of asymptomatic infection,namely male(OR=1.73,95%CI=1.05?2.85),living/working outdoor(OR=1.96,95%CI=1.05?3.65),and the greater travelling distance every week(1?5km vs.?1km:OR=2.76,95%CI=1.55?4.92;>5km vs.?1km:OR=2.88,95%CI=1.21-6.87)were risk factors.Compared with genotypes AA,the genotype AG of TLR9 T1486C was protective factor for asymptomatic malaria infections(cOR=0.45,95%CI=0.24?0.84).The analysis of molecular marker polymorphism associated with drug resistance showed that proportion of mutant molecular marker in P.vivax asymptomatic infections was considerable,including 100%Pvdhfr mutant type,40%Pvdhps mutant type,100%Pvmdrl mutant type,40%Pvcrt-o mutant type,while Pfcrt mutant type was also found in P.falciparum asymptomatic infections.There was no significant difference between the distribution of molecular marker between asymptomatic and symptomatic malaria.The cohort study found that only 3.85%(3/78)asymptomatic infection became symptomatic malaria during the follow up.Kaplan Meier analysis showed that the median duration of asymptomatic infections was 8 weeks.There were several patterns of oscillations of asymptomatic infections including persistent asymptomatic(24.36%),positive followed by negative(20.51%),intermittent positive(32.05%),only positive at cross-sectional survey(11.54%).Age,frequent travelling and forest exposure history were the important factors affecting the duration of asymptomatic infections by Cox proportional risk regression model.Compared with the group of<20 years old,the probability of asymptomatic infections from positive to negative was higher in the group of>20 years old,HR is 2.11(9 5%CI=1.12?3.97);compared with the group without travelling,probability of asymptomatic infections from positive to negative for frequent travelers were lower,HR was 0.39(95%CI=0.16?0.96);compared with those without forest exposure,the probability of asymptomatic infections with forest exposure from positive to negative was lower,HR was 0.51(95%CI=0.27?0.94).Conclusions:The prevalence of asymptomatic malaria infections was high along the China-Myanmar border.It was statistically significantly higher in Myanmar sites than that Yunnan site in China,which indicated the similar trend with incidence of symptomatic infections.Asymptomatic malaria infection was epidemiologically characterized by greater prevalence in male and 51-60 yr age group.The age-specific prevalence of asymptomatic infections was different between male and female.Gender,main living/working place and greater travelling distance every week were the main influencing factors of asymptomatic infections.The epidemiological characteristics showed that asymptomatic infections were closely associated with human mosquito exposure,which was similar with symptomatic malaria.The cohort study found that nearly one quarter of asymptomatic infections were persistent,with median duration of 8 weeks.Age,travelling frequency and forest exposure history were the main factors related to the duration of asymptomatic infections.