In the present population-based study on Flores Island, Indonesia, P. falciparum, P. vivax, P. malariae and mixed infections were detected with PCR in 11.4%, 10.5%, 1.5%, and 3.1% of the participants, respectively. Only 87% of all infections were revealed using real-time PCR. The higher sensitivity of the real-time PCR is reflected in the lower DNA load in microscopy-negative subjects compared to the DNA load found in microscopy-positive subjects.
Plasmodium falciparum prevalence increased up to 15–19 years of age and decreased in the older age groups; also the parasite load was significantly higher in the participants under 20 years whereas the prevalence of P. vivax started to decrease somewhat earlier in age. In areas with higher transmission intensity, such as Irian Jaya and in Africa, the peak in prevalence and parasitaemia is usually observed at a younger age due to an earlier acquired immunity [22–24].
Although only significant for P. vivax, the numbers of malaria infections were found higher in males than females. This could be more due to working outdoors in the field resulting in a higher exposure to mosquito bites, but then higher P. falciparum and P. malariae infections would be expected. This was also shown in a study performed in India  but in contrast with a study in Popondeta, Papua New Guinea, located close to the eastern part of Indonesia, where no significant difference was found in malaria prevalence between males and females . A more detailed statistical analysis of links between gender, work, local ecological, environmental, economic and cultural factors, which might influence exposure to risk of infection and the disease, has been planned for the future.
Clearly anaemia is a well-known phenomenon associated with malaria disease, but also in the present study among apparently healthy individuals, anaemia was strongly correlated with the prevalence and load of P. falciparum infections, which is in agreement with other studies in asymptomatic subjects [27–32]. In the present study, anaemia was also correlated with the load of P. vivax infections. Although anaemia is a known complication in cases with severe P. vivax malaria, it is less known in latent P. vivax cases and might be underestimated due to misdiagnosis. Obviously anaemia is a multifactorial clinical phenomenon  and no correction was performed for potential other causes.
Several studies were performed to understand the contribution of ABO blood group antigens in protection against Plasmodium infections. Although meta-analysis has shown that blood group O provides selective advantages against severe malaria, other studies were unable to link ABO blood groups to the incidence of malaria [34–37]. In the present cross-sectional study, the prevalence of P. falciparum and P. vivax was higher, although not significantly in subjects with blood group AB compared to subjects with the other blood groups. This seems in contrast with previous findings; however, in the present study mostly sub microscopic Plasmodium infections were found using real-time PCR whereas previous studies used microscopy to detect malaria parasites. Further studies in areas of different endemicity using Plasmodium PCR will be needed to elucidate the role of ABO blood groups in acquiring infection and the outcome of disease in malaria.
The real-time PCR procedure as described in this paper is not suitable to use as a point of-care (POC) test in “treatment on diagnosis” control strategies. The high-throughput, however makes it very appealing to transport samples to a central laboratory for monitoring the effect of mass drug administration programs . Moreover, nucleic acid based POC assays are in development as well, such as LAMP (loop-mediated isothermal amplification), nucleic acid amplification protocols directly from blood without DNA extraction, and lateral flow detection of PCR products [39–42].
In the present study real-time PCR has proved to be a powerful diagnostic tool providing novel insights into the epidemiology of malaria infections in a low transmission area. The high number of sub microscopic infections have important implications in the transmission dynamics [43–48] and have to be considered in novel elimination strategies [38, 49–51]. Moreover, these low density malaria infections appear to play a role in eliciting or maintaining humoral immune responses [48, 52, 53] and therefore should be taken into account in, for example studies on the immunology in malaria infections and the association with the regulatory responses that may be caused by chronic worm infections interacting with inflammation .