To guide strategic planning for the transition from malaria control to elimination, we utilized novel malaria surveillance techniques of pooled PCR and serology to perform the first large scale cross-sectional malaria survey in Swaziland and found that prevalence of infection and recent exposure was extremely low. By pooled PCR, only one P. falciparum and one P. malariae infection were identified among 4028 participants. Compared to RDT, pooled PCR identified infections missed by RDT and provided improved efficiency and affordability excluding capital costs. Serological data identified a potential focus of recent transmission in the southeast and low seroprevalence in younger age groups, suggesting low exposure to malaria in recent years. Report of recent travel to Mozambique was identified as a risk factor for both P. falciparum infection and seropositivity.
Large-scale prevalence surveys have traditionally relied on microscopy, but considering the significant time and labor required, and potential operational limitations, the Swaziland malaria program decided to use RDTs. As a simple point-of-care test, RDTs are convenient, but can give false positive results with underlying autoimmune conditions, non-malarial infections, or persistence of the P. falciparum
HRP-2 antigen despite resolution of infection 
. Pooled PCR is extremely specific, 100%, because repeat testing of the sample at each stage limits DNA contamination 
. Using pooled PCR as gold standard, there were three false positives by RDT in our study, and due to the extremely low prevalence, positive predictive value was poor. With large-scale surveys in higher prevalence settings, RDT have also had low positive predictive value 
RDTs may also miss infections of low parasite density and many do not detect non-falciparum species 
. In our study, the P. falciparum
-specific RDT missed one P. malariae
infection, and one P. falciparum
infection that was likely of low parasite density given that the participant was afebrile. Others have found low parasite density to be a determinant of decreased sensitivity in survey settings 
. The detection limit of current RDTs is 100–200 parasites/µL. The detection limit of pooled PCR can reach submicroscopic levels but sensitivity is only reliable at 100 parasites/µL 
. It is possible that the three RDT positives were true infections missed by pooled PCR. As an antigen-based assay, RDT could potentially be more sensitive if there is sequestering of P. falciparum
parasites, particularly in low density infections. These subjects could have also had a recent infection that cleared, but persistent antigenemia. While not assessed in this study, microscopy and individual PCR are more sensitive than pooled PCR 
. However, the efficiency provided by pooled PCR, and potential for improved sensitivity over RDT, suggest that for large-scale surveys among asymptomatic populations in low prevalence settings, pooled PCR may be preferred. Compared to performing individual PCR testing, use of pooling in this study reduced labor and consumable costs for PCR by greater than 95%.
One limitation with measuring parasitemia in a cross-sectional survey is that regardless of the sensitivity of the test, only one moment in time is captured. In low endemic settings such as Swaziland, prevalence may be so low that it will be difficult to track progress toward elimination. As a measure of past infection, serology has been proposed as a useful way to estimate exposure in low endemic settings 
. In our study, a statistically significant difference in seroprevalence and seroconversion rate among participants less than 20 years of age compared to older participants suggests a significant decrease in transmission in 1989, with low stable levels of transmission since that time 
. Declining incidence in Swaziland over the past 20 years is consistent with this finding 
Serological data may additionally help to identify other epidemiological risk factors for malaria. Travel to Mozambique and residence in the southeastern region were found to be associated with seropositivity. The first finding is consistent with Swaziland's passive surveillance and case investigation data, which have found travel to Mozambique to be a risk factor for infection 
. The second finding was unexpected as incidence in the southeastern region of the country has been low in recent years 
. However, there are many sugar plantations in this area and migrant workers from Mozambique may be a source of transmission. Our data did not include information on occupation or nationality, but suggest that detailed, focal investigation of this region is indicated to determine if seropositivity represents past infection acquired in Mozambique or local transmission.
One potential limitation in our study is that cutoff for seropositivity was based on an assumption of a bi-modal distribution of seropositives and seronegatives within the population sampled. Samples from representative seronegatives (e.g. age- and genetically-matched participants from Swaziland with no history of falciparum exposure but similar exposure to other infections that could result in cross-reactive antibodies) were not available and in practice may be difficult to obtain. Our data suggest a clear temporal trend of a decline in malaria transmission 20 years prior. However, the potential non-specific response seen among younger age groups may suggest that this method is not specific enough to show cessation of transmission, as has been shown in other settings 
. To enable more accurate estimates of further decrease in transmission using serology, longitudinal studies are needed to better characterize the development, maintenance, and decay of specific antibodies 
Findings of this study have important implications on Swaziland's strategic planning for malaria elimination. First, the findings of extremely low parasite prevalence and recent exposure suggest that the high IRS coverage (45% compared to other parts of Africa, where IRS coverage is approximately 10% 
) and the cross-border collaboration with Mozambique and South Africa have been effective and should be continued. Second, low transmission in spite of low ITN use suggests that further investment into this costly program may not be justified. Rather, ITNs could be reserved for use in identified hot spots and high-risk groups. Third, limited access to diagnostic and treatment services among febrile participants identified passive surveillance and case management as an area for improvement. Finally, future efforts should aim to prevent imported malaria and investigate and target interventions to limited foci of transmission.
For low transmission countries aiming to eliminate malaria, reliance on microscopy or RDT for active surveillance may be inadequate. We document the first national survey from an elimination setting to show that pooled PCR and serology, as accurate and efficient methods to measure current and past infection, can provide critical data to inform strategic planning. Additionally, renewed interest in elimination has been accompanied by skepticism about the feasibility of elimination, particularly in sub-Saharan Africa 
. This study documents an extremely low prevalence of malaria infection and recent exposure in Swaziland, providing evidence-based optimism for efforts in Swaziland and the region.