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Gaining an in-depth understanding of malaria transmission requires integrated, multifaceted research approaches. The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) is applying specifically developed molecular and immunological assays, in-depth entomological assessments, and advanced statistical and mathematical modeling approaches to a rich series of longitudinal cohort and cross-sectional studies in Papua New Guinea and Cambodia. This is revealing both the essential contribution of forest-based transmission and the particular challenges posed by Plasmodium vivax to malaria elimination in Cambodia. In Papua New Guinea, these studies document the complex host–vector–parasite interactions that are underlying both the stunning reductions in malaria burden from 2006 to 2014 and the significant resurgence in transmission in 2016 to 2018. Here we describe the novel analytical, surveillance, molecular, and immunological tools that are being applied in our ongoing Asia-Pacific ICEMR research program.

The Asia-Pacific International Center of Excellence in Malaria Research (ICEMR) was funded in 2016 to conduct a coordinated set of field and in-depth biological studies in Cambodia and Papua New Guinea (PNG), in sites that span the range of transmission intensities currently found in the Asia-Pacific regions. The overall objective is to gain an understanding of key parasite, human host, and vector factors involved in maintaining transmission in the face of intensified control and elimination programs, and to develop novel approaches to identify and target residual transmission foci. In this article, we will describe how the ICEMR program was designed to address key knowledge gaps and priority areas for the malaria control programs in each country. In PNG, partners have worked together on two consecutive ICEMR grants (2009–2016 and 2017–2024) and we present a case study of the partnership and engagement approach that has led to stronger coordination of research activities and integration with program, informing country-level strategic planning and prioritization of control activities. In both settings, the ICEMR program has generated insights into transmission foci, risk factors for ongoing transmission, highlighting the hidden burden of vivax malaria, and the need for additional complementary vector control tools. Finally, we will summarize the emerging research questions and priority areas—namely surveillance, vivax malaria, new vector control tools, and community/health systems-oriented approaches—where further tool development and implementation research have been identified as being needed to guide policy.

The authors recently reported that long-lasting insecticidal nets (LLINs) distributed in Papua New Guinea (PNG) between 2013 and 2019, exhibited severely diminished efficacy to knock down and kill susceptible Anopheles mosquitoes. This coincided with a rise in malaria observed in PNG since 2015. Here, the authors show that LLIN bioefficacy is increased by heating LLINs prior to WHO cone bioassays. Unused LLINs with low bioefficacy, delivered to PNG in 2019, were heated to 120°C for 5 minutes. Cone bioassays were performed before and at 1 hour, 7 days, and 30 days after heating. This led to a significant increase in 24-hour mortality (17–61%) and 60-minute knock down (31–72%). The effect was sustained over 30 days. Bioassays are crucial in quality assurance of LLIN products. Our findings indicate that bioefficacy of LLINs can be increased by heating. This may have implications for quality assurance procedures used to assess LLINs.

An important aspect of many malaria molecular epidemiology and transmission studies is RNA-based detection of gametocytes. Ensuring RNA stability represents a challenge in tropical, resource-limited environments, as RNA may quickly degrade when samples are not preserved under adequate conditions. This study investigated the degradation of pfs25 messenger RNA (mRNA), the most widely used Plasmodium falciparum gametocyte marker, in whole blood spiked with cultured P. falciparum gametocytes, exposed to different temperatures for up to 48 hours, and collected with different anticoagulants. The levels of pfs25 mRNA were similar between samples stored at 4°C and 30°C for up to 48 hours before stabilization with RNAprotect (Qiagen, Hilden, Germany). We observed that pfs25 mRNA in heparin-collected blood degraded less than that in ethylenediaminetetraacetic acid (EDTA)–collected blood over the 48-hour period. For field studies aiming for P. falciparum gametocyte detection, immediate stabilization of blood samples is not necessary, as the pfs25 transcript is relatively stable, more so in heparin than EDTA collection tubes.

We conducted a prospective longitudinal study of fetal size in rural Papua New Guinea (PNG) involving 439 ultrasound-dated singleton pregnancies with no obvious risk factors for growth restriction. Sonographically estimated fetal weights (EFWs; N = 788) and birth weights (N = 376) were included in a second-order polynomial regression model (optimal fit) to generate fetal weight centiles. Means for specific fetal biometric measurements were also estimated. Fetal weight centiles from a healthy PNG cohort were consistently lower than those derived from Caucasian and Congolese populations, which overestimated the proportion of fetuses measuring small for gestational age (SGA; < 10th centile). Tanzanian and global reference centiles (Caucasian weight reference adapted to our PNG cohort) were more similar to those observed in our cohort, but the global reference underestimated SGA. Individual biometric measurements did not differ significantly from other cohorts. In rural PNG, a locally derived nomogram may be most appropriate for detection of SGA fetuses.

Malaria control can be improved by rapid, sensitive, low-cost detection of infection. Several such strategies are being pursued. Rapid diagnostic tests can detect infections at parasite densities above 200 μL(-1). Polymerase chain reaction methods can detect low parasite densities, but are slow and prone to contamination under field conditions. Methods that detect hemozoin presence in blood have been proposed as alternatives for rapid detection of infection. In this study, we used a benchtop nuclear magnetic resonance (NMR) device to detect hemozoin. This device could be deployed in malaria-endemic settings. We measured synthetic hemozoin in phosphate-buffered saline and malaria parasites in human blood. The NMR detected hemozoin in suspensions of 4 ng μL(-1) and parasites at densities of 8,000-10,000 μL(-1) (0.2% parasitemia). Thus, our preliminary NMR approach, although providing very rapid measurements, is unlikely to achieve the required sensitivity and specificity for malaria diagnosis, unless a preliminary concentration step is performed.

A method of gametocyte quantitation in human blood was developed based on magnetic fractionation using commercially available magnetic fractionation columns (MFCs) and exploiting the magnetic susceptibility of mature Plasmodium falciparum gametocytes. The technique uses magnetic microspheres as a calibration standard. Microspheres are added to each blood sample to a known concentration. When exposed to a magnetic field, gametocytes and magnetic microspheres are preferentially captured inside MFCs. After removal of the magnetizing field, the magnetically captured material can be eluted, placed on a microscope slide that is stained, and counted by using conventional methods. The limits of quantitation for P. falciparum gametocytes were determined from serial dilutions of blood samples with known gametocyte density. The upper limit was 1,000 gametocytes/μL. Quantitative analysis above this threshold is difficult because of an over-abundance of gametocytes. The lower limit was 0.1 gametocytes/μL, and there is a significant probability of a false-negative result below this level.