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Table 1 Summary of the three main diagnostic methods in childhood malaria with their characteristic features

From: Diagnostic tools in childhood malaria

Method Key characteristics Parasite species detectable Advantages Disadvantages Reference
Microscopic diagnosis Conventional bright field microscopic examination Giemsa-stained thick blood film to determine the presence or absence of malaria parasite Plasmodium genus-specific - Small amount of sample (blood) is required;
- Able to quantify parasitaemia;
- Provides prognostic information that serves as indicator for disease severity (morphological characteristics of the parasites, the maturity of asexual stages of the parasite);
- Cost effective compared to molecular techniques
- Difficulties in detecting parasites in low parasite density samples (50–100 parasites/μl);
- Malaria microscopist expert/well-trained personnel is needed to interpret the result (high morphological similarities between P. falciparum, P. malariae and P. knowlesi could lead to misdiagnosis and treatment delay)
[5, 13, 14, 33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48]
Giemsa-stained thin blood film to identify the Plasmodium species All human Plasmodium species
Quantitative buffy coat method (QBC) Detection of malaria parasites in centrifuged peripheral blood by staining the parasite DNA with acridine orange and examination under fluorescence microscope Plasmodium genus-specific - Higher sensitivity (5 parasites/μl) compared to bright field microscopic examination;
- Fast and easy to be performed;
- Interpretation of the result is simple and requires less-trained personnel
- Difficulties in parasite species differentiation and subjective parasite quantification;
- Specific equipment (fluorescence microscope) is required
[49,50,51,52,53,54,55,56,57]
Partec Rapid Malaria Test (PT) Detection of malaria parasites using test slide that is readily labelled with 4′-6-diamidino-2- phenylindole (DAPI) which binds to intraerythrocytic Plasmodium DNA, resulting in fluorescence under Partec CyScope® (fluorescence microscope) Plasmodium genus-specific - Easy and rapid, less labour-intensive and requires less training time for laboratory personnel;
- Could be used in the field without electricity supply;
- Small amount of sample (few μl) is required
- Difficulties in species differentiation;
- False positive results due to the presence of non-specific artefacts or nuclei-containing cells (reticulocytes, leukocytes and bacterial cells);
-Specific equipment (Partec CyScope®) is required
[58,59,60]
Rapid diagnostic test (RDT) OptiMAL® Detection of malaria via the pLDH antigen P. falciparum and P. vivax Ease of use, rapid diagnosis and result interpretation, sensitive, field-deployable Less sensitive compared to molecular diagnostic methods, heat sensitive, reduced sensitivity for non-falciparum malaria, false-negative results due to low-level expression or deletion of target antigen genes (pfhrp2) [83]
ParaSight-F test Detection of malaria via the HRP-2 antigen P.falciparum [84]
Immunochromatographic test (ICT) Malaria PF test Detection of malaria via the HRP-2 antigen P.falciparum [84]
SD Bioline Malaria AG Pf/Pan Detection of malaria via the HRP-2 and pLDH antigen P. falciparum (HRP-2), pan-malarial (pLDH) [85]
CareStart™ Malaria Detection of malaria via the HRP-2 and pLDH antigen P. falciparum (HRP-2), pan-malarial (pLDH) High specificity and PPV Low sensitivity at low parasite densities [87]
Malaria pf Rapid device Detection of malaria via the HRP-2 antigen P. falciparum Sensitivity and specificity comparable to those for light microscopy [88]
Ultra sensitive RDT (uRDT) Detects HRP-2 antigen of P. falciparum malaria P. falciparum Higher sensitivity, specificity and ability to detect new infections faster than conventional RDT Similar to conventional RDTs, is less sensitive compared to molecular diagnostic methods [89]
Molecular diagnostic methods Nested PCR Targeting 18S rRNA gene Plasmodium genus-specific Elevated sensitivity compared to RDTs and microscopy Cumbersome, expensive, and requires well-trained staff with stringent laboratory cleanliness to minimize risk of contamination [101]
Targeting 18S rRNA gene Plasmodium genus-specific followed by nested species-specific PCR More sensitive than microscopic examination for identification of asymptomatic malaria [108]
Targeting cytochrome b gene Plasmodium genus-specific Detection limit of 10 parasites/μl, better than single-round PCR and real-time methods [41, 104, 105, 107]
Semi-nested PCR Targeting 18S rRNA gene P. falciparum and P. vivax More sensitive than microscopic examination for identification of sub-microscopic infections [103]
Quantitative nucleic acid sequence-based amplification (QT-NASBA) Targeting 18S rRNA gene. Quantification was achieved by co-amplification of the RNA in the sample with one modified in vitro RNA as a competitor P. falciparum - Fast, sensitive, reliable, and quantitative;
- Allowed for the sub-microscopic quantification;
- Detection limit of 10 parasites/μl
[101, 102]
Multiplex PCR Targeting 18S rRNA gene P. falciparum and P. vivax - Detection limit of 0.1 parasites/μl;
- No cross-reaction between Plasmodium spp.;
- Able to detect the mixed infection
[106]
Real-time quantitative PCR (qPCR) Targeting plasmepsin 4 in P. falciparum and the aspartic protease PM4 in P. vivax P. falciparum and P. vivax - Quantification of parasite densities;
- More sensitive than microscopic examination;
- Detection limit of 5.6 copies/μl;
- Able to detect and quantify infections that have very low infection (0.001%)
[103]
qPCR Targeting telomere-associated repetitive element 2 and the var. acidic terminal sequence P. falciparum - Detection limit of 0.03 to 0.15 parasites/μl;
- 10× more sensitive than standard 18S rRNA qPCR
[110]
Reverse transcription-polymerase chain reaction (qRT-PCR) Targeting 18S rRNA P. falciparum and P. vivax Able to detect and differentiate submicroscopic malaria infections as low as 10 parasites/ml and 18 copies/μl [111]