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Sustained Malaria Control Over an 8-Year Period in Papua New Guinea: The Challenge of Low-Density Asymptomatic Plasmodium Infections

Continuous malaria control in Papua New Guinea has resulted in a marked decline of Plasmodium falciparum and P. vivax prevalence. Yet, an increasing proportion of submicroscopic infections, many of them carrying gametocytes, demands for novel strategies to target residual transmission.

the 2 species, and in many countries P. vivax has proven more resilient to control [5,12,13]. P. vivax densities determined by microscopy are generally 5-10 times lower than P. falciparum densities [14][15][16], making diagnosis more difficult. Latent liver-stage parasites (hypnozoites) escape diagnosis, and standard treatment against blood-stage parasites does not affect them [17]. In regions where malaria is highly endemic, up to 80% of all blood-stage P. vivax infections in children are due to relapses [18,19]. If the transmission level declines, individuals who have experienced high levels of transmission may harbor a large reservoir of hypnozoites, which will result in relapses for an extended period. Thus, the proportion of all blood-stage parasite infections in the population that are caused by relapsed P. vivax relapses as compared to primary infections might temporarily increase.
Few in-depth studies have assessed the effect of intensified control on parasite prevalence, clinical malaria, the proportion of asymptomatic and submicroscopic P. falciparum and P. vivax infections, and gametocyte carriage over several years in the same population. In the Madang area on the north coast of Papua New Guinea (PNG), P. falciparum and P. vivax prevalence determined by polymerase chain reaction (PCR) analysis had reached 30%-60% in the general population during 2001-2006 [14,16,[20][21][22]. As a consequence of the corresponding high transmission intensity, children in PNG acquired natural immunity against clinical malaria during early childhood, and 78%-97% of infections in the general population were asymptomatic [14,16]. In 2008/2009 and again in 2011/2012, long-lasting insecticidal nets were distributed in PNG. Rapid diagnostic tests to test all febrile cases in health centers, as well as artemisinin-based combination therapy with artemether-lumefantrine as first-line treatment, were implemented in 2012. Surveys conducted after the first round of long-lasting insecticidal net distribution found considerable decreases in entomological inoculation rate [23] and parasite prevalence detected by LM [24], suggesting these interventions had a pronounced effect on transmission.
To understand the full impact of intensified control, repeated cross-sectional surveys were conducted in Madang Province in 2006, 2010, and 2014 ( Figure 1). Blood samples were collected from 5913 individuals, and highly sensitive molecular assays were used to diagnose malarial infections and gametocytes in the same population during distinct phases of malaria control.

Ethics Statement
Informed written consent was obtained from participants, or, if participants were <18 years, from their parents or legal guardians.

Study Site and Sample Collection
Blood samples were collected in Madang Province (Figure 1), in coastal catchments for 2 health centers (Mugil and Malala), and 1 inland catchment (Utu). The climate is tropical, with a rainy season from December to April. Samples were collected during March-April in 2006 and between mid-May and early July during 2010 and 2014. A convenience sampling strategy including individuals aged >6 months was used. In 2014, among villages, 8.3%-45.1% of residents were sampled (Supplementary Table 1).
From each participant, a 250-μL blood sample obtained by finger prick was collected into ethylenediaminetetraacetic acidlined tubes. For gametocyte detection, 50 μL of blood was transferred into tubes containing 250 μL of RNAprotect (Qiagen; performed during 2010 and 2014 only). In the field, samples were stored at 4°C and transferred every night to the laboratory at −20°C (for DNA extraction) or −80°C (for RNA extraction).

Parasite Quantification By qPCR and LM
Laboratory methods described elsewhere were used [25]. In brief, DNA was extracted from 200 μL of pelleted blood, using the Favorgen 96-well genomic DNA extraction kit and eluted in 200 μL of buffer. P. falciparum and P. vivax, as well as Plasmodium malariae and Plasmodium ovale (during the 2010 and 2014 surveys only), were quantified by highly sensitive qPCR assays, using 4 µL of DNA, corresponding to 4 µL of blood [26]. A dilution of plasmids containing the target sequence of the PCR was run as an external standard for absolute quantification. P. falciparum-positive samples were genotyped by msp2 [22,27], and P. vivax-positive samples were genotyped by msp1F3 and MS2 [28,29].
For gametocyte detection, RNA was extracted using the Qiagen RNeasy 96-kit, with additional DNAse treatment to remove residual DNA (Qiagen RNase-Free DNase Set). pfs25 and pvs25 transcripts were detected using published reverse-transcription qPCR protocols [30] and were quantified using plasmids to generate an external standard curve. A genus-specific qPCR assay [30] was run to ensure absence of DNA.

Data Analysis
Data were analyzed using Stata 12.1. Unless otherwise stated, results are based on qPCR analysis. Densities (determined by LM or qPCR analysis) are given as geometric means. Fever or history of fever was defined as measured fever >37.5°C or reported febrile illness in the past 2 days. Clinical malaria was defined as fever or history of fever and detection of parasites by microscopy. Logistic regression was used to assess risk factors of infection, and χ 2 tests were used to compare rates of infection between age groups and catchments. Only in rare cases was an individual included in >1 survey, because the 3 surveys did not necessarily involve the same villages. These cases were treated as independent observations.

Model for Age-Prevalence Curves
The nonlinear association between parasite prevalence and age was first assessed using generalized additive models with thinplate smoothing splines, for each survey. The shift in age-prevalence peaks across surveys was then investigated using a likelihood-based model inspired by the work of Smith et al [31]. The host population was described using a compartmental model similar to that of a classical SIRS model, using a set of 3 ordinary differential equations (ODEs). Instead of modeling the proportions of susceptible (s k ), infected (i k ), and retired (r k ) individuals in survey k according to time, these were modeled according to age a: Hence, this ODE model did not represent actual transmission events but rather provided an estimate of age-prevalence curves. A binomial likelihood function was used to fit the model to A likelihood ratio test was used to assess the statistical significance between the null and alternate models.  Mean P. falciparum and P. vivax gene copy numbers decreased 10-fold (P < .001) and 5-fold (P < .001),  respectively, from 2006 to 2014 (Table 1 and Figure 2B). In all surveys, the mean P. falciparum density was 5-10fold higher than the mean P. vivax density. As a result of lower parasite densities, the proportion of submicroscopic infections increased between 2006 and 2014, from 37.2% to 72.1% for P. falciparum (P < .001) and from 62.0% to 86.7% for P. vivax (P < .001; Table 1). A generalized additive model indicated that in response to increased training, LM-based diagnosis had become more sensitive over time (Supplementary Figure 1). Assuming identical LM sensitivity in all 3 surveys, the increase in the proportion of submicroscopic infections would have been even more pronounced. Densities of both species decreased with age. The decrease in P. falciparum densities was slower in 2010 and 2014 as compared to 2006 (interaction of log 10 age with log 10 density: P = .042; Figure 4A). This effect was even more pronounced for P. vivax, with little change of densities with age in 2014 (P < .001; Figure 4B). By genotyping, a pronounced increase in the proportion of single-clone infections was observed. The proportion of P. falciparum single-clone infections was 57.0% in 2006, 80.1% in 2010, and 82.3% in 2014 (P < .001). For P. vivax, the proportions were 50.9%, 61.3%, and 78.7% in 2006, 2010, and 2014, respectively (P < .001).

Parasite Prevalence and Density
In multivariable analysis, age was highly associated with the risk of infection in all surveys and for both species (Supplementary Table 3). The P. falciparum prevalence differed between catchments in all surveys, but not the P. vivax prevalence. Treatment with antimalarials in the past 2 months resulted in an approximately 50% reduction of the odds of P. falciparum or P. vivax infection in 2006 (P < .001). No such association was observed in 2010 and 2014 (P ≥ .079).

Clinical Symptoms
Improvements in morbidity indicators were observed over the 8-year period ( Table 2). The proportion of individuals who reported having experienced a malaria episode in the past 2 weeks or who had received antimalarials in the previous 2 months decreased 12-fold and 6-fold, respectively (P < .001 for both comparisons; Table 2). The proportion of qPCR-positive infections defined as clinical malaria decreased 2-3-fold (P ≤ .039; Table 2), and the population attributable fraction of fever or history of fever caused by LM-positive infections decreased substantially (P < .001; Table 2). In contrast, measured fever did not change significantly (P = .843).
The proportion of participants presenting with an enlarged spleen decreased from 30.2% to 1.3% ( ; in 2014, this association was even stronger (OR, 7.99; P < .001). The proportion of participants with moderate-to-severe anemia (defined as a hemoglobin level of <8 g/dL) halved between 2006 and 2014 (P < .001; Table 2).
In 2014, P. falciparum prevalence was 4.7% in Utu as compared to 8.6% and 12.3% in Mugil and Malala, respectively (P < .001). At the village level, substantial P. falciparum spatial heterogeneity was observed (Supplementary Table 1). In 9 villages, P. falciparum prevalence was low (range, 0%-5.6%), while in 8 villages, it ranged from 8.3% to 22.2%. The diversity of parasite populations remained high, even when prevalence was low. In 6 of the low-prevalence villages, ≥2 isolates were genotyped by pfmsp2, and within each village, different clones were detected (Supplementary Table 4).

DISCUSSION
Along the north coast of PNG, continuous control of malaria over 8 years has led to a 12-and 6-fold decrease of P. falciparum and P. vivax prevalence, respectively, detected by LM. Using a highly sensitive qPCR to diagnose infections, the continuous decrease in P. falciparum prevalence was confirmed, whereas the P. vivax prevalence increased between 2010 and 2014. Parasite densities of both species have decreased considerably, and thus an increasing proportion of infections were asymptomatic and submicroscopic.
Gametocyte densities and the probability to detect gametocytes-and, thus, human-to-mosquito transmission potentialwere closely correlated to blood-stage parasite density. Because of the lower parasite densities, gametocytes were detected in a lower proportion of infections in 2014 than in 2010. However, because of the increase in the proportion of submicroscopic infections, remaining gametocyte carriers became more difficult to identify. For both species, the majority of gametocyte carriers (determined by reverse-transcription qPCR) were LM positive for asexual parasites in 2010, but in 2014 approximately two thirds of gametocyte carriers presented with submicroscopic infections. Over 90% of gametocyte carriers were asymptomatic, and such individuals thus present a challenge for malaria control and elimination. In PNG, most febrile cases presenting at health centers are diagnosed by LM or rapid diagnostic test and antimalarial treatment is given to positive individuals. The increasing proportion of asymptomatic and submicroscopic infections thus remain untreated, yet such infections of both P. falciparum and P. vivax have been shown to frequently infect mosquitos [8][9][10].
Decreasing levels of transmission also appeared to have an impact on acquisition of immunity to P. falciparum. In malaria-endemic countries, the attack rate in children increases with age [32], and in parallel individuals acquire immunity gradually, with the speed of acquisition depending on the transmission intensity. As a result, clinical malaria and parasite prevalence peaks in children and then decreases as immunity is acquired [33]. In 2006, very high parasite densities were observed in young children, followed by a rapid decline with increasing age. This age-associated decline was less marked in 2014, and the peak P. falciparum prevalence shifted from children to adolescents, reflecting delayed acquisition of immunity, similar to trends observed for clinical malaria in Africa [34]. In parallel, the odds of presenting with fever when infected with P. falciparum increased 4-fold between 2006 and 2014, further suggesting a reduced level of (clinical) immunity. Individuals with low levels of immunity are expected to present with higher parasite densities, and the risk of developing clinical malaria increases. However, over the 8-year period, parasite densities for both species decreased considerably, and the proportion of individuals with clinical malaria decreased 3-fold. Parasite densities are determined not only by acquired immunity, but also by the age of the infection on the day of sampling (Supplementary Figure 2). Densities in the peripheral blood peak in the first phase of the infection, and when not treated they can persist for weeks or months at low densities [3]. When transmission is lower, fewer new infections are acquired and persisting infections are thus on average older and densities lower. For example, the molecular force of P. vivax bloodstage infection in PNG children decreased from 15 clones/year [35] to 5 clones/year between 2006 and 2010 [19]. In 2014, the contribution of older low-density infections appeared to be far greater than the contribution of infections with possibly higher initial parasite densities caused by lower levels of immunity. In addition, 6-fold less treatment was administered in 2014 as compared to 2006, further contributing to a large number of old, low-density chronic infections.
The overall decrease in prevalence was accompanied by increasing heterogeneity of P. falciparum prevalence at the village level, indicating foci of residual transmission. In contrast to clonal P. falciparum outbreaks observed in the highlands of PNG [36], Solomon Islands [37], and South America [38], the parasite populations in the 2014 survey remained genetically diverse, even in villages with very low prevalences. This could indicate that residual infections were imported from villages with higher transmission, where a genetically diverse population is maintained.
In contrast to the constant decline in P. falciparum prevalence, P. vivax prevalence has increased since 2010. In PNG, in 2008-2010, 80% of all blood-stage P. vivax infections in children were caused by relapses [18,19]. As a consequence, for P. vivax, mosquito-to-human transmission levels and parasite prevalence rates in the population are less correlated than for P. falciparum. In 2014, P. vivax densities were very low, and almost 80% were single-clone infections. Both factors suggest high proportions of relapses. Relapses often consist of a single clone [39][40][41], and often consist of clones that are homologous or related to the initial blood-stage parasite infection [40,42]. They thus carry a reservoir of antigens the immune system has been exposed to recently, and even young children with limited acquired immunity may be able to control such infections [43]. An increasing proportion of infections caused by relapses of previously acquired infections could thus explain the low P. vivax densities across all ages in 2014 and the increase in P. vivax prevalence from 2010 to 2014. It is possible that the 2014 survey captured a phase during which the vast P. vivax hypnozoite reservoir that had accumulated in the population from years of high transmission had not yet been exhausted and that the prevalence had thereby increased temporarily.
In conclusion, the rapid decline in transmission in a population maintaining a relatively high level of clinical immunity resulted in a large proportion of very-low-density infections. Increasing proportions of submicroscopic infections when prevalence is lower has been found across countries both for P. falciparum and P. vivax [3,4]. Few studies have assessed the speed of change in the same population. In the Brazilian Amazon, a 9-fold decrease of P. vivax prevalence over 3 years was accompanied by an increase in the proportion of submicroscopic infections, from 44% to 73%, and almost all of them carried gametocytes [12]. The present finding of an increasing proportion of gametocyte carriers being submicroscopic-despite an overall lower proportion gametocyte positive-is thus likely a general pattern in countries where transmission levels are decreasing.
Novel strategies are therefore needed to effectively target the asymptomatic low-density reservoir of Plasmodium infections. For P. falciparum, mass screen and treat (MSAT) approaches would require highly sensitive diagnostics tools. For P. vivax, in which hypnozoite carriers cannot be detected with any current diagnostic test, MSAT is not an appropriate intervention [19], and other approaches will need to be developed. The large asymptomatic and submicroscopic reservoirs thus represent a challenge to the goal of a malaria-free Asia-Pacific for the foreseeable future. An in-depth understanding of their contribution to maintaining transmission and better tools and surveillance strategies to efficiently identify and target these infections are thus urgently needed.

Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes
Disclaimer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Financial support. This work was supported by the International Centers of Excellence for Malaria Research (grant U19 AI089686), the TransEPI consortium, funded by the Bill and Melinda Gates Foundation; the NHMRC (grant 1021544; early career fellowship 1016443 to L. J. R.); the Victorian State Government, through operational infrastructure support; the Australian Government NHMRC IRIISS; the Swiss National Science Foundation (fellowship P2BSP3_151880 to C. K.); the Medical Research Council (population health scientist fellowship to M. W.); and the NHMRC (senior research fellowship to I.M.).
Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.