Study settings and participants

A cross-sectional study was conducted in Ingapu Township, Myanmar, during the rainy season in August 2018. This region was previously characterized by the Myanmar National Malaria Control Programme as a medium-transmission area, with reported usPCR prevalence of 8.7% P. falciparum infections and 5.6% Plasmodium vivax infections during the rainy season in 2015 [11]. A study carried out in 2015 found the annual parasite index (API) in the region to be 1–5 malaria cases per 1000 individuals under surveillance per year [2]. Individuals aged 6 months or older, including pregnant women and the elderly, were enrolled in the study after provision of written informed consent. Parents/guardians provided written consent for participants younger than 18 years old. Written assent was also provided by children age 8 to 17 years of age. Enrollment in the study was offered to individuals attending a mobile clinic staffed by local public health volunteers. One hundred individuals were enrolled.

To identify potential markers of asymptomatic usPCR+ malaria infections, a retrospective case-control study nested within a previously reported 2015 cross-sectional survey [11] in which participants provided consent for additional analysis of samples was performed. Samples originated from Injanyang and Ann Townships, both of which have reported API > 5 cases per 1000 individuals under surveillance per year [2]. No participants reported malaria symptoms, nor were any RDT-positive. Antibody responses were measured from archived case and control DBS samples on Protein Saver 903 Cards. Cases (n = 50) were individuals who tested positive for P. falciparum by usPCR, and controls (n = 175) were individuals who tested negative for P. falciparum by usPCR. This study was designed specifically to identify antibody markers of usPCR+ P. falciparum malaria infections, so individuals with P. vivax mixed and mono-infections were included in the case and control groups, respectively, to account for potential cross-reactive responses. The case group included 25 individuals with mixed P. falciparum and P. vivax infections. The control group included 25 individuals who were P. vivax-positive.