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Additional file 2. Parasite Density Estimation.

Additional file 3. EasyScan Go.

Additional file 1. STARD-2015-Checklist.

BackgroundMass administrations of antimalarial drugs (MDA) have reduced the incidence and prevalence of P. falciparum infections in a trial in the Greater Mekong Subregion. Here we assess the impact of the MDA on P. vivax infections.MethodsBetween May 2013 and July 2017, four villages in each Myanmar, Vietnam, Cambodia and Lao PDR were selected based on high prevalence of P. falciparum infections. Eight of the 16 villages were randomly assigned to receive MDA consisting of three-monthly rounds of three-day courses of dihydroartemisinin-piperaquine and, except in Cambodia, a single low-dose of primaquine. Cross-sectional surveys were conducted at quarterly intervals to detect Plasmodium infections using ultrasensitive qPCR. The difference in the cumulative incidence between the groups was assessed through a discrete time survival approach, the difference in prevalence through a difference-in-difference analysis, and the difference in the number of participants with a recurrence of P. vivax infection through a mixed-effect logistic regression.Results3,790 (86%) residents in the intervention villages participated in at least one MDA round, of whom 2,520 (57%) participated in three rounds. The prevalence of P. vivax infections fell from 9.31% to 0.89% at month 3 but rebounded by six months to 5.81%. There was no evidence that the intervention reduced the cumulative incidence of P.vivax infections (95% confidence interval [CI] Odds ratio (OR): 0.29 to 1.36). Similarly, there was no evidence of MDA related reduction in the number of participants with at least one recurrent infection (OR: 0.34; 95% CI: 0.08 to 1.42).ConclusionMDA with schizontocidal drugs had a lasting effect on P. falciparum infections but only a transient effect on the prevalence of P. vivax infections. Radical cure with an 8-aminoquinoline will be needed for the rapid elimination of vivax malaria.

Malaria rapid diagnostic tests (RDTs) primarily detect Plasmodium falciparum antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for P. vivax and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens. A multiplexed immunoassay for the quantification of HRP2, P. vivax LDH, and all-malaria LDH (pan LDH) was developed to accurately measure circulating antigen concentration and antigen distribution in a population with endemic malaria. The assay also measures C-reactive protein (CRP) levels as an indicator of inflammation. Validation was conducted with clinical specimens from 397 asymptomatic donors from Myanmar and Uganda, confirmed by PCR for infection, and from participants in induced blood-stage malaria challenge studies. The assay lower limits of detection for HRP2, pan LDH, P. vivax LDH, and CRP were 0.2 pg/ml, 9.3 pg/ml, 1.5 pg/ml, and 26.6 ng/ml, respectively. At thresholds for HRP2, pan LDH, and P. vivax LDH of 2.3 pg/ml, 47.8 pg/ml, and 75.1 pg/ml, respectively, and a specificity ≥98.5%, the sensitivities for ultrasensitive PCR-confirmed infections were 93.4%, 84.9%, and 48.9%, respectively. Plasmodium LDH (pLDH) concentration, in contrast to that of HRP2, correlated closely with parasite density. CRP levels were moderately higher in P. falciparum infections with confirmed antigenemia versus those in clinical specimens with no antigen. The 4-plex array is a sensitive tool for quantifying diagnostic antigens in malaria infections and supporting the evaluation of new ultrasensitive RDTs.

Additional file 1: S1 Table. Definition of primary efficacy endpoint by initial species. S2 Table. Dose of each compound given to the participants. S3 Table. The baseline characteristics of women with Plasmodium falciparum mono-infection. S4 Table. The baseline characteristics of women with Plasmodium vivax mono-infection. S5 Table. The baseline characteristics of patients with Plasmodium falciparum infection, including both P. falciparum mono-infection and co-infection of P. falciparum and Plasmodium vivax. S6 Table. Cumulative proportion of fever and parasite clearance of falciparum and vivax for each treatment arm among pregnant women with history of fever or documented fever at enrolment. S7 Table. Description of gametocyte carriage, congenital and placental malaria. S8 Table. Univariable and multivariable analyses of the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model. S9 Table. Univariable and multivariable analyses on the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model with Pfkelch13 mutations. S10 Table. Prevalence of symptoms before and after treatment. S11 Table. Description of pregnancy outcomes. S12 Table. Details of pregnant women who had miscarriage or stillbirth. S13 Table. Details of congenital abnormality and ICD-10 coding. S14 Table. Details of neonatal mortality. S15 Table. Univariable and multivariable linear regression analyses of the characteristics associated with gestational week at birth among those who had malaria in pregnancy. S16 Table. Univariable and multivariable linear regression analyses of the characteristics associated with SGA z-score (birthweight for gestational age and newborn sex at birth). S17 Table. Birth outcomes in pregnant women who had malaria and were enrolled in the first trimester. S1 Figure. Monthly number of pregnant women with malaria for each malaria species over the study period (from February 2010 to August 2016). S2 Figure. Kaplan-Meier survival curves for antimalarials in pregnancy: Panel a. recurrence of any malaria species, Panel b. Plasmodium falciparum PCR-corrected efficacy censoring intercalated Plasmodium vivax malaria, Panel c. P. falciparum PCR-uncorrected efficacy , and Panel d. P. vivax parasitaemia after P. falciparum mono-infection. S3 Figure. Kaplan-Meier survival curves for parasite clearance of Plasmodium falciparum (Panel a) or Plasmodium vivax (Panel b) for each treatment arm. S4 Figure. Fractional change in haematocrit from baseline during the follow-up for each species (Panel a) or treatment stratified by malaria species (Panel b).

Additional file 1: S1 Table. Definition of primary efficacy endpoint by initial species. S2 Table. Dose of each compound given to the participants. S3 Table. The baseline characteristics of women with Plasmodium falciparum mono-infection. S4 Table. The baseline characteristics of women with Plasmodium vivax mono-infection. S5 Table. The baseline characteristics of patients with Plasmodium falciparum infection, including both P. falciparum mono-infection and co-infection of P. falciparum and Plasmodium vivax. S6 Table. Cumulative proportion of fever and parasite clearance of falciparum and vivax for each treatment arm among pregnant women with history of fever or documented fever at enrolment. S7 Table. Description of gametocyte carriage, congenital and placental malaria. S8 Table. Univariable and multivariable analyses of the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model. S9 Table. Univariable and multivariable analyses on the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model with Pfkelch13 mutations. S10 Table. Prevalence of symptoms before and after treatment. S11 Table. Description of pregnancy outcomes. S12 Table. Details of pregnant women who had miscarriage or stillbirth. S13 Table. Details of congenital abnormality and ICD-10 coding. S14 Table. Details of neonatal mortality. S15 Table. Univariable and multivariable linear regression analyses of the characteristics associated with gestational week at birth among those who had malaria in pregnancy. S16 Table. Univariable and multivariable linear regression analyses of the characteristics associated with SGA z-score (birthweight for gestational age and newborn sex at birth). S17 Table. Birth outcomes in pregnant women who had malaria and were enrolled in the first trimester. S1 Figure. Monthly number of pregnant women with malaria for each malaria species over the study period (from February 2010 to August 2016). S2 Figure. Kaplan-Meier survival curves for antimalarials in pregnancy: Panel a. recurrence of any malaria species, Panel b. Plasmodium falciparum PCR-corrected efficacy censoring intercalated Plasmodium vivax malaria, Panel c. P. falciparum PCR-uncorrected efficacy , and Panel d. P. vivax parasitaemia after P. falciparum mono-infection. S3 Figure. Kaplan-Meier survival curves for parasite clearance of Plasmodium falciparum (Panel a) or Plasmodium vivax (Panel b) for each treatment arm. S4 Figure. Fractional change in haematocrit from baseline during the follow-up for each species (Panel a) or treatment stratified by malaria species (Panel b).