The PCR analyses done here helped to establish two relationships, i.e. the rate of false positives and false negatives given by microscopy and RDT, respectively. Both have implications for malaria control measures. Starting with the false positive rate and its implications, in the present study, anti-malarial drugs were prescribed for 9.3 and 20.0% microscopy- and RDT-based parasite-negative patients, respectively, as witnessed by PCR. This percentage of parasite-negative patients receiving anti-malarial drugs was comparable to countries such as Tanzania
 and Zanzibar
 that have high malaria transmission settings. The percentage of false positive patients (parasite-negative patients) receiving anti-malarials in the study under report was much lower than patients from low-moderate malaria transmission areas in Tanzania (63.0%)
. In this study, in five microscopy-positive samples (three P. falciparum and two mixed infections) all of which were determined as P. falciparum, PCR failed to detect them. They were considered false microscopy positives since PCR was considered the reference. Of the RDT positives, PCR failed to detect 20 samples, all of which were determined as P. falciparum, by RDT and hence called false RDT positives. Among many other factors, the utility of a RDT-based negative result depends on the sensitivity of the test
, storage conditions (>30°C), and low levels of parasitaemia
For false negatives, the discussion spins around 19.2% prevalence of sub-microscopic P. falciparum. The results of this study indicate that considerable numbers of P. falciparum infections were missed by both RDT and microscopy, suggesting the need for a more sensitive assay for the detection of sub-microscopic parasitaemia. There was no difference in sub-microscopic carriages in febrile and non-febrile patients (OR = 0.83; P = 0.872). The identification of sub-microscopic P. falciparum infection in fever patients may not help to reliably confirm malaria as the cause of the fever and excludes the possibility of other diseases
. The presence of sub-microscopic asymptomatic P. falciparum infections may represent a significant challenge to malaria control programmes since such parasitaemic individuals may serve as a reservoir of infection and contribute to mosquito infection
[24–26]. Moreover, a mass screening and treatment campaign may include such individuals, but their parasitaemia would remain invisible to the classical light microscopy or RDT and this necessitates the use of molecular screening techniques. Studies have indicated that microscopy misses on average half of all P. falciparum infections in endemic areas compared to PCR
. The agreement between RDT and microscopy was 89.4% (42/47) and 71.4% (five of seven) in detecting P. falciparum and mixed infections, respectively. Both RDT and microscopy underestimated the true parasite prevalence in the study area. In this study, microscopy and RDT detected 38.9 and 60% of the infection identified by PCR.
The limitations of commercially available RDT kits are well documented
[27, 28]. The false positivity in RDT may arise due to circulating PfHRP2 antigen from recent infections
[5, 29]. Because RDTs that detect HRP-2 antigen cannot distinguish between active infections and resolved infections. RDTs based on detection of the HRP-2 antigen often remain positive for over five weeks after the disappearance of live parasites, because they detect the HRP-2 antigen which is still present in debris from dead parasites for some time after total parasite clearance
. In this study, false positivity rate of RDT was 2.1% higher than microscopy. The false positive rate of RDT in this study was fairly in agreement with previous evaluations of RDTs in population-based household surveys among healthy persons in Ethiopia
, 1.5%, but much lower than reported in Zambia
, 7.9%, when compared with microscopy. Such false positive readings in malaria diagnostic tests will overestimate the true parasite prevalence compared with expert microscopy. In spite of the apparent low sensitivity of RDT compared to PCR in this study, whether any deletion of HRP2-antigen exists among P. falciparum in Ethiopia requires further research.
The identification of large numbers of sub-microscopic parasitaemia in this study was supported by meta-analysis that demonstrates high proportion of sub-microscopic infection of P. falciparum in areas of low transmission
, indicating that those with little previous exposure are able to control parasite densities. The presence of high proportion of sub-microscopic infections could indicate a recent decrease in transmission in Ethiopia and this is consistent with data from northern Tanzania showing high (33%) prevalence of almost entirely sub-microscopic infections during a time of declining transmission
. In a study conducted in Cambodia, microscopy detected a total of 350 P. falciparum infections while PCR detected a further 331 P. falciparum infections from microscopy-negative samples
. The importance of sub-microscopic parasitaemia in sustaining the parasite population has been indicated
. PCR also has a limit of detection; the number of sub-microscopic infections is likely higher than reported in this study.
The prevalence of microscopic P. falciparum infection decreases with age since younger age groups have the highest incidences as determined by microscopy, RDT and PCR. On the other hand, the proportion of sub-microscopic P. falciparum infections was higher in older age groups. This is because increasing age has been clearly linked to lower parasite densities
 and most infections in older children and adults are sub-microscopic compared with young children
. Children present with symptomatic malaria at a younger age in areas of high transmission than in areas with lower transmission
. Under conditions of very low transmission, the risk of clinical disease extends into adulthood
 where risks of a clinical event are more directly related to the risks of infection than the effect of acquired clinical immunity. But an increase in the prevalence of sub-microscopic P. falciparum infection with age may explain the fact that infections will be controlled and remain asymptomatic in older age groups because clinical immunity develops overtime.
The observation of high prevalence of PCR-based sub-microscopic P. falciparum in the study area has important implications for malaria control measures in Ethiopia since such infections are important contributors to the infectious reservoir
[36–38], because, even at low densities, these infections could be a potential source of transmission for vectors
[26, 33] and a potential source of malaria attack within the population. Several studies have indicated that the contribution of sub-microscopic parasites to malaria transmission in individuals was similar to those individuals having microscopic gametocytes
 while others indicated that sub-microscopic carriers were several times less infectious than the microscopic carriers
. For assessing progress in reducing malaria transmission, PCR seems the best tool for the estimation of parasite prevalence in the general population.
In this study, older age was associated with increased sub-microscopic carriages. This finding is in agreement with a study by Manjurano and his colleagues
 where individuals older than 15 years were three times more likely to have sub-microscopic parasites than younger individuals. It is apparent that the risk of malarial infection could be reduced by bed net use. In this study, use of bed nets was not found to be protective against sub-microscopic carriage. This is supported by a study where 50% of net users from high transmission areas were PCR positive compared to 75% of non net users at the lowest altitude
. The high incidences of sub-microscopic carriages observed in older age groups as compared to the younger age group (<15 years) in this study is supported by another study
 conducted in Uganda. Moreover, Vafa and her colleagues
 have reported that sub-microscopic carriage differs in relation to transmission intensity and age. Nevertheless, asymptomatic carriage is probably a common occurrence in the study area. Further studies to unveil the magnitude of sub-microscopic asymptomatic carriage will be indispensable for guiding and monitoring future elimination efforts in Ethiopia.