This study evaluated the performance of different diagnostic tools in detecting malaria infection among febrile children attending outpatient clinic at Korogwe District. PCR performed better than blood slide expert microscopy and HRP-2-based malaria RDT. Overall, malaria prevalence was low and significantly associated with age where the majority of patients were over 12 months old. Infants appear to be relatively protected from malaria (and other infections) for the first 6 months of life, mainly due to acquiring maternal antibodies and the presence of foetal haemoglobin [
22‐
24]. Three-quarters of malaria patients had low levels of parasitaemia (<200,000 parasites/μL).
The use of HRP-2-based malaria RDT in the current study resulted in over-diagnosis of malaria infection in 15 % of patients. This might suggest the presence of recent malaria infection. When compared with expert microscopy and PCR, the sensitivity value of malaria RDT was below the ≥95 % threshold recommended by WHO. These findings are similar to those reported by Shakely and colleagues on a hospital-based study conducted in Zanzibar, an area of low malaria transmission [
25]. HRP-2-based malaria RDT is known for detecting malaria antigens that continue to circulate in blood almost 2 months after treatment of a malaria episode [
26]. This is the most probable explanation for the false-positive results and sub-microscopic infections that were observed in the study. In the current study, eight patients had false-negative malaria RDT results despite having moderate to high levels of parasitaemia. This could be due to malaria parasites expressing low level of target antigen, deleted
pfhrp2 gene, prozone effect or other factors, which could not be established [
27‐
32]. However, current information from WHO indicates that in most settings, genetic mutations (deletion of
pfhrp2/
pfhrp3) in parasites are not likely to be the main cause of false-negative results [
33]. Therefore, further research is required to determine the true prevalence of these mutations. Despite these limitations, HRP-2-based malaria RDTs remain the preferred choice, mainly in settings with limited microscopy facilities, due to easy availability and low cost [
34]. There are continued efforts to improve sensitivity and specificity of malaria RDTs for rapid and better management of malaria cases. Clinicians are advised to investigate other causes of febrile episodes despite a positive malaria RDT test. Different types of infections are common in children from resource-limited areas that lack adequate sanitation and clean water supply. Therefore, it is very likely that children attending outpatient clinics from rural settings present with multiple infections [
35].
Identification of sub-microscopic infection by PCR has demonstrated that expert microscopy can miss detection of malaria in patients having low parasite densities hence leading to false-negative results. As malaria transmission declines, cases of sub-microscopic infection in both symptomatic and asymptomatic persons is likely to increase [
36]. PCR and other molecular techniques are indicated to be the most sensitive diagnostic tool than microscopy and RDT [
37,
38]. However, its utility in routine practice remains a concern especially at this era towards malaria elimination.
The study had a number of limitations. Firstly, some of the patients may have been on anti-malarial therapy despite verbal confirmation provided by their parents/guardians on the prior use of anti-malarial drugs. This would have contributed to the false-positive RDT results. Secondly, the malaria RDT and PCR diagnostic tools used in the current study could not detect other species of
Plasmodium apart from
P. falciparum. This could have missed a few malaria infections of other species, such as
P. malariae and
P. ovale that have been reported in the country [
29,
39,
40]. Both
P. malariae and
P. ovale occur more commonly as mixed infections with
P. falciparum [
40‐
42]. Lastly, the study may have biased the estimated prevalence of sub-microscopic infections due to the unavailability of nearly a quarter of patient samples for PCR analysis.