Ethiopia includes regions of differing malaria endemicity and transmission. The peak of malaria incidence follows the main rainfall season (July-September) each year. However, many areas in the south and west of the country have a rainfall season beginning earlier in March and May or have no clearly defined rainfall season . Futhermore, the sample collection was carried out during the dry season in this area.
A SnM-PCR was considered and used in this study as a molecular gold standard. The prevalence obtained by SnM-PCR was 7%, higher than the estimated by LM method, which demonstrates an increased sensitivity with respect to the latter. In this regard, it is noteworthy that all microscopy-positive samples were also found to be positive by SnM-PCR. However, the SnM-PCR test was able to detect positive samples that were missed by microscopy.
In this study a high number of children (80%), who initially had been negative for LM method, were later found to be infected with P. falciparum by SnM-PCR. These undetected sub-microscopic infections do not develop the disease in the patients but they may have an enormous impact on malaria transmission in endemic areas. Infected individuals may play the role of parasite reservoir since their blood is able to infect mosquito vectors and they may reintroduce malaria into certain regions [26, 27]. Molecular methods have potential use to detect malaria parasites in asymptomatic infections, mainly in children infected with P. falciparum, which causes the most severe malaria infection.
The qPCR sensitivity was 70% when compared to SnM-PCR. Despite measurements taken over 45 cycles in qPCR, previous studies and the manufacturers consensus rule was considered to decide whether a sample was positive or not (Ct value <40 ).
The qPCR is the most analytically sensitive method (sensitivity 70%), followed by microscopy (sensitivity 52.5%), when compared to SnM-PCR. Overall, qPCR shows substantial agreement with other molecular techniques for the detection of Plasmodium prevalence. The SnM-PCR has been reported for the diagnosis of low-level parasitaemia, and real-time PCR for the diagnosis of high-level parasitaemia. In countries where malaria is not endemic and where it has recently re-emerged, there is lack of physicians and microscopy-skilled laboratory staff specializing in malaria, which makes the diagnosis of malaria difficult. Therefore, SnM-PCR is a good alternative since it does not require special training for interpretation of the results, the opposite to microscopy, in which specific training is needed for species differentiation. In addition, SnM-PCR is not altered by the subjectivity of the observer .
The advantage of qPCR to other molecular techniques is the quantification of parasite densities. The correlation between quantifications by both qPCR showed a high correlation (R2 = 0.980) confirming that qPCR using commercial kits (PrimerDesignTMLtd) is an accurate method to quantify parasite densities. When correlating quantification by qPCR with LM counts in samples where both techniques showed positive results, a low correlation for P. vivax (R2 = 0.586) was found. This low value might be due to overall lower densities, possibly around the detection limit in P. vivax infections, because the scarcity of the template in case of a very low parasite density is expected to lead to imperfect detection . Another possible explanation would be the presence of schizonts in the blood, because clinical samples with similar levels of parasitaemia by LM but with different proportions of schizonts (14–21 genomes) will vary in copy number and consequently in real-time PCR parasite quantification. In this sense, the comparison of parasitaemia determined by LM with that assessed by genomic standard DNA quantification curve showed significantly divergent results among malaria samples .
Microscopic diagnosis is considered to be the reference standard for determining the protective efficacy of prophylactic drugs or vaccines. However, microscopy is an imperfect reference standard with many inherent limitations . The difficulties in correctly identifying low-density infection may contribute to the discrepancy in microscopy results. Furthermore, these discrepancies indicate a need to implement a rigorous quality assurance system within the routine laboratory diagnostic techniques for malaria in Ethiopia. Molecular techniques, such as PCR, have a lower detection threshold for Plasmodium than microscopy [30, 31], and it may be a more sensitive diagnostic tool in a population where low-density infections are expected. For this reason, the study addressing malaria situation in Gambo was carried out using SnM-PCR.
The prevalence observed was 7%, which is higher than the prevalence observed in previous surveys performed in Amhara (4.1%), Jimma (5.2%) and SNNP Regional States (5.4%) [32–34]. This is due to the fact that the prevalence value was obtained with SnM-PCR, which is a more sensitive technique than microscopy. The prevalence of Plasmodium species was 69.7% and 30.3% for P. vivax and P. falciparum, respectively, unlike the previous paradigm of Plasmodium species composition in Ethiopia (P. falciparum 60% and P. vivax 40% of total malaria cases) . These differences seem to indicate that the proportion of both species is changing. A study, carried out from 2007 to May 2009. It is also important to highlight the fact that this study was carried out with patients admitted to a hospital in a rural area and thus this was a biased study. In the area of study a decrease in cases of P. falciparum mono-infection and an increase of P. vivax mono-infection have been previously reported  and another study, conducted in areas of Oromia in December 2009, found a greater proportion of Plasmodium infection due to P. vivax. The result of the present study, carried out between January and May 2010, may be explained due to the tendency of P. vivax to cause long-term chronic infection  and to dominate during low transmission periods and dry season (March-June) .
In the present study, a significant association between children patients and number of positive blood smears (χ
2 = 5.93 p < 0.05) was found. Where transmission is relatively stable and intense, the mean and median age of patients diagnosed with malaria at clinics is generally under five years old . For this reason, due to such age-dependency of the infection, children acquire certain malaria immunity . Studies reported that individuals living in areas of unstable and low intensity malaria transmission do not acquire significant immunity to the disease, and hence malaria infections can be observed in all age groups [41, 42]. In the present study, the least affected age group was the group over 30 years old (9.1%). The epidemiological condition prevailing in Gambo from a prospective parasitological survey point of view suggests that the area is characteristic of a stable, moderate level of malaria endemicity being considered a meso-endemic area, based on the classification established by WHO .