Community-based molecular and serological surveillance of subclinical malaria in Myanmar

In the Greater Mekong Subregion (GMS), current malaria surveillance strategies rely on a network of village health volunteers (VHVs) reporting the results of rapid diagnostic tests (RDTs), known to miss many asymptomatic infections. Integration of more sensitive diagnostic molecular and serological measures into the VHV network may improve surveillance of residual malaria transmission in hard-to-reach areas in the region and inform targeted interventions and elimination responses. However, data on residual malaria transmission that would be captured by these measures in the VHV-led testing and treatment surveillance network in the GMS is unknown.

Methods
A total of 114 VHVs were trained to collect dried blood spots from villagers undergoing routine RDTs as part of VHV-led active and passive case detection from April 2015 to June 2016. Samples were subjected to molecular testing (quantitative polymerase chain reaction [qPCR]) to determine Plasmodium falciparum and P. vivax infection and serological testing (against P. falciparum and P. vivax antigens) to determine exposure to P. falciparum and P. vivax.

Results
Over 15 months, 114 VHVs performed 32,194 RDTs and collected samples for molecular (n = 13,157) and serological (n = 14,128) testing. The prevalence of molecular-detectable P. falciparum and P. vivax infection was 3.2% compared to the 0.16% prevalence of Plasmodium spp. by RDT, highlighting the large burden of infections undetected by standard surveillance. Peaks in anti-P. falciparum, but not P. vivax, merozoite IgG seroprevalence coincided with seasonal P. falciparum transmission peaks, even in those with no molecularly detectable parasites. At the individual level, antibody seropositivity was associated with reduced odds of contemporaneous P. falciparum (OR for PfCSP 0.51 [95%CI 0.35, 0.76], p = 0.001, PfAMA1 0.70 [95%CI 0.52, 0.93], p = 0.01, and PfMSP2 0.81 [95%CI 0.61, 1.08], p = 0.15), but not P. vivax infection (OR PvAMA1 1.02 [95%CI 0.73, 1.43], p = 0.89) indicating a potential role of immunity in protection against molecular-detectable P. falciparum parasitaemia.

Conclusions
We demonstrated that integration and implementation of sample collection for molecular and serological surveillance into networks of VHV servicing hard-to-reach populations in the GMS is feasible, can capture significant levels of ongoing undetected seasonal malaria transmission and has the potential to supplement current routine RDT testing. Improving malaria surveillance by advancing the integration of molecular and serological techniques, through centralised testing approaches or novel point-of-contact tests, will advance progress, and tracking, towards malaria elimination goals in the GMS.

Peer Review reports
Background
The emergence of artemisinin-resistant Plasmodium falciparum within the Greater Mekong Subregion (GMS) has led to the region setting elimination targets for all human malaria by 2030 [1]. In working towards this goal, the incidence of malaria cases and deaths in the GMS fell substantially by 75% and 93%, respectively, between 2012 and 2017 [2]. Monitoring and surveillance are critical to the elimination of malaria ensuring that progress towards malaria elimination targets can be accurately tracked and ultimately accelerated. However, as the region transitions towards malaria elimination, surveillance becomes increasingly difficult because malaria becomes concentrated in discrete geographical foci, such as border and hard-to-reach areas, and in high-risk populations such as migrant workers and residents of highly forested areas [3,4,5]. To capture these infections, in many remote areas, surveillance and malaria control strategies are dependent on passive case detection (PCD) and active case detection (ACD) provided by a village health volunteer (VHV) network who administer malaria testing by rapid diagnostic test (RDT) as well as treatment. However, subclinical Plasmodium spp. infections often go undetected because asymptomatic individuals are less likely to seek testing and treatment and, importantly, are generally below the detection limit of conventional RDT diagnostics used in the field. Undetected, and therefore untreated, Plasmodium spp. infections may be an important source of residual malaria transmission [6,7,8,9,10], and failure to detect and eliminate all infections may hinder malaria elimination