Study sites and design
The studies were conducted in two Provinces on the highly endemic north coast region of Papua New Guinea (PNG) (Figure 1). In the WHO Western Pacific Region (WPR), the malaria mortality rate declined by 58% over the period 2010–2015, however infection prevalence in Papua New Guinea (PNG) remains the highest in this region (and outside the African continent), contributing 81% of malaria cases and 86% of malaria deaths in 2017 in the region (WHO, 2017, 2018) primarily due to P. falciparum and P. vivax infections (Kattenberg et al., 2020; Koepfli et al., 2017; WHO, 2018). In 2003, a new national malaria control campaign was launched to achieve high levels of LLIN ownership and usage in PNG (Hetzel et al., 2014; Hetzel et al., 2012). Coverage with LLIN was low in most parts of the country before nationwide free distribution took place (2004-8 and 2009-2012) (Betuela et al., 2012; Genton et al., 1994; Hii et al., 2001). This resulted in a significant increase in ownership of bed nets across the country by 2010 (any type 80%; LLINs 65%) (Hetzel et al., 2014; Hetzel et al., 2012) and the average malaria incidence rate in sentinel sites dropped from 13/1,000 population to 2/1,000 (range 0.6-3.3/1000 post-LLIN) (Hetzel et al., 2016).
On the hyperendemic north coast, P. falciparum PCR prevalence dropped from 38% to 12% and P. vivax prevalence decreased from 28% to 13% (Kattenberg et al., 2020; Koepfli et al., 2017) (Figure 1). Prevalence reductions were more substantial for P. falciparumthan for P. vivax, as has been seen in many co-endemic areas (Feachem et al., 2010). In East Sepik Province, malaria decreased from a very high burden (73% of surveyed individuals infected in 2005 as measured by molecular detection (LDR-FMA (Mueller et al., 2009) to heterogeneous transmission (prevalence in villages ranging from 1% to 61%, median 6%, as measured by qPCR (Kattenberg et al., 2020)) after two rounds of LLIN distribution. An initial round of LLIN distribution was conducted between 2004 and 2009, followed by additional distributions in 2011/2012 and subsequently in 2014/15. In Madang province, however, malaria prevalence decreased from 63% to 28% by qPCR after the first round of LLIN distributions (Koepfli et al., 2017; Koepfli et al., 2015; Schultz et al., 2010). After the second LLIN distribution (2010-2014), P. falciparum continued to drop, however an increase in P. vivax prevalence was observed (from 13% to 20% by qPCR (Koepfli et al., 2017)). In Madang province, malaria prevalence was less heterogenous in the sampled villages than in East Sepik Province (Kattenberg et al., 2020; Koepfli et al., 2017; Koepfli et al., 2015).
Whole blood samples were collected from participants in cross-sectional studies conducted between 2005 and 2014 along the North Coast of PNG (Figure 1) (Arnott et al., 2013; Kattenberg et al., 2020; Koepfli et al., 2017; Koepfli et al., 2015; Mueller et al., 2009; Schultz et al., 2010). In Madang Province (MAD), the same three catchment areas were studied in 2006 (Schultz et al., 2010; Senn et al., 2012), 2010 (Koepfli et al., 2015) and 2014 (Koepfli et al., 2017). The study area included a selection of villages along a coastal stretch of 70km (Mugil and Malala regions), surrounded by coconut and cocoa plantations and subsistence gardens, and one area approximately 50 km inland (Utu). Here, the climate is tropical with a rainy season from December to April. In East Sepik Province (ESP), participants in the Wosera Catchment (ESP1) including fourteen villages were sampled in 2005 during the dry-season (August-September) (Jennison et al., 2015; Senn et al., 2012). A broader survey (ESP2) was conducted in April-May 2005 including five catchment areas that were re-visited in 2012-13 (Kattenberg et al., 2020; Mueller et al., 2009). The study areas in ESP consist of an area of over 160 km2 with low hills and riverine plains with a wet, tropical climate (Genton et al., 1995). The natural vegetation is lowland hill forest that has mostly been replaced by re-growth following cultivation and wide grasslands on the plains near the Sepik River.
In all surveys, demographic and clinical information was collected, blood slides examined by expert microscopists and a blood sample collected in EDTA tubes for extraction of DNA. In the 2005 ESP studies,Plasmodium species were detected by Light Detection Reaction- Fluorescent Microspere Assay (LDR-FMA) (McNamara et al., 2006; Mueller et al., 2009), whereas in all other studies quantitative PCR (qPCR) detection by TaqMan™ assay was used (Rosanas-Urgell et al., 2010). To determine multiplicity of infection (MOI), P. falciparum positive samples were genotyped for Pfmsp2 and P. vivax positive samples were genotyped with Pvmsp1f3 and MS16 (ESP1 and MAD 2006) orPvmsp1f3 and MS2 (MAD 2010 & 2014 and ESP2 2005 & ESP 2012-13) ), as previously described (Arnott et al., 2013; Kattenberg et al., 2020; Koepfli et al., 2017; Koepfli et al., 2015; Mueller et al., 2009; Schultz et al., 2010). The sample selection and genotyping procedures of the ESP1 2005 (Wosera) and MAD 2006 were as previously described (Arnott et al., 2013; Jennison et al., 2015; Schultz et al., 2010). For the other studies, samples with MOI of 1 were selected for further genotyping with the neutral microsatellite panels as described below. For the studies conducted after the large scale LLIN distribution (>2006) all monoclonal isolates (MOI=1) were included, but for the 2005 ESP2 population, a selection of samples was made for the analysis with the microsatellite panel (Table S1).