Background
The Global Technical Strategy for Malaria of the World Health Organization (WHO) calls for a world free of malaria [
1]. To contribute to this global vision and encouraged by the substantial gains made in malaria control over the last two decades, Ethiopia has embarked on progressively eliminating malaria starting from low malaria transmission areas [
2‐
6]. In the initial phase, 239 woredas (districts) were targeted for malaria elimination by the National Malaria Control and Elimination Programme of Ethiopia [
5]. Preparations are underway to shift diagnosis and surveillance approaches from reducing malaria morbidity and mortality to detecting infections and measuring transmission in the selected woredas [
2,
5].
Malaria elimination requires the detection and clearing of all
Plasmodium infections. A relatively high prevalence of subpatent or low parasite density infections, that are often missed by conventional diagnostic methods (microscopy and rapid diagnostic tests) are being reported especially from low transmission settings [
7‐
14]. Such asymptomatic, subpatent infections could be explained by acquired immunity in higher transmission settings [
15]. Even in low transmission settings, asymptomatic and subpatent infections might play a role in transmission dynamics, hindering the progress of malaria elimination [
14‐
19].
In Ethiopia,
Plasmodium falciparum and
Plasmodium vivax are the major reported malaria parasite species with rare reports of
Plasmodium malariae and
Plasmodium ovale. In 2017, out of a total confirmed malaria cases of 1,530,739,
P. falciparum accounted for 1,059,847 cases and
P. vivax for 470,892 cases [
1,
2].
The current study assessed the magnitude of subpatent infections in northwestern Ethiopia, using samples collected in 2015 from a nationwide household survey of malaria endemic areas.
Discussion
In this study, results of microscopy, RDT and nPCR detection methods were used to examine the prevalence of
Plasmodium infection in malaria endemic settings in northwest Ethiopia. Nested PCR identified 30 additional positive malaria infections that were missed by RDTs in the field, and thus not provided treatment at the time of the survey. The prevalence reported by nPCR was 3.3%, 16.1% higher than that detected by RDTs and 64.4% higher than from microscopy. More than 60% of the nPCR positive cases were identified from Benishangul-Gumuz Region, which is one of the higher malaria transmission region in Ethiopia [
5]. The region showed a similar high prevalence of malaria by microscopy and RDTs in this study and in a separate serology study of the same samples [
21].
The study compared microscopy and RDT detection methods with a pooling nPCR approach that can detect parasite density as low as 0.1–10 parasites/µl of blood [
22‐
26]. Close agreement was observed between both RDT and microscopy results compared to PCR with 80.8% and 83.9% concordance, respectively. Microscopy, considered the gold standard for malaria diagnosis [
26], has a variable limit of detection (50–100 parasite/µl of blood) [
26‐
28], depending on the skill of the technician and the reagents used [
26‐
28]. As per the national malaria diagnosis and treatment guideline [
29], Ethiopia uses microscopy in health centres and hospitals and RDTs in health posts (primary health care units). In the current study with slide preparation and RDTs conducted under field conditions where test requirements may not be optimal, high variability was observed in the results across the three methods. This discordance becomes disproportionately higher in these low transmission settings with very few positive samples. The strong association between seropositivity and PCR positivity suggests a role for using multiplex seroprevalence results to more rapidly and inexpensively identify hotspots or confirm lack of
Plasmodium infection in areas reporting low incidence.
Although the lower RDT positivity is likely due to low-density infections, the possibility of hrp2/3 gene deleted
P. falciparum infections has been raised in Ethiopia with alarming reports originating from Eritrea [
29‐
33]. Although HRP2 and HRP3 deletions were reported recently from a study in Amhara [
34], a study of larger geographic scope is currently ongoing in Ethiopia (Sindew Mekasha, EPHI, personal communication).
Several studies have reported asymptomatic
Plasmodium infections in low transmission settings using PCR from a range of countries [
7‐
15,
18,
22,
35,
36]. The terms asymptomatic, submicroscopic, and subpatent are often used interchangeably to describe a malaria infection that cannot be detected by conventional methods (microscopy and RDTs) but can be detected by more sensitive methods such as PCR. The prevalence of subpatent infection in low transmission setting has ranged from 0.003 to 44%, depending on the sensitivity of the tools used and the sample collection area [
36]. Studies in Iran [
37] and Sri Lanka [
38] reported zero prevalence by the more sensitive methods, indicative of no local transmission and confirmation of malaria elimination. Golassa et al. [
14] and Tadesse et al. [
15] reported PCR prevalence ranging from 1.7 to 5.8% in southwest Ethiopia, comparable to the 3.3% prevalence reported in the current study.
Subpatent infections, despite the low parasite density, could be infectious to mosquitoes [
15,
19,
39]. Studies in The Gambia [
40], Thailand [
35], Peru [
41] and Ethiopia [
17] showed even low-density, asymptomatic infections could be infectious to mosquitoes, which may pose a potential, unidentified reservoir for malaria transmission. A recent review [
19] summarized that lower density of parasites were seen in low transmission compared to high transmission settings and argued that subpatent infections contribute to the infectious reservoir, could be long-lasting, and predictive of future periods of patent infections.
Several new tools are being developed with higher limits of parasite detection in field settings, such as, ultrasensitive RDTs and loop-mediated isothermal amplification (LAMP) [
35,
42]. The current study used a sample pooling methodology that decreases cost and time and is applicable to screening large number of samples, particularly from low transmission settings [
22,
43]. The low blood volume eluted from the DBSs in the current study may limit parasite detection as the volume of blood analysed is critical in determining its limit of detection [
44,
45]. Although more sensitive molecular methods have been developed [
46], the cost and feasibility of the tests in field settings could limit their wide-scale adoption and uptake in resource-limited countries.
Acknowledgements
We acknowledge the MIS 2015 Steering Committee, data collectors and study participants for the samples collected. We thank Zewditu Bekele, Fitsum Tesfaye, Hilina Legesse and Hailemariam Difabachew from the Ethiopian Public Health Institute, for sample organization and management.
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