The present study assessed the performance of SDFK90 for malaria diagnosis, a RDT detecting both PfHRP2 and Pf-pLDH, using stored samples obtained in international travellers suspected of malaria. Overall sensitivity for P. falciparum was 98.5% and reached 99.3% at parasite densities above 100/μl. Specificity for non-falciparum and Plasmodium negative samples was 98.8%.
A number of limitations need to be considered. Due to the use of stored samples, clinical information potentially explaining false positive or false negative results, such as the visible PfHRP2 line among three P. vivax samples, was not available. Furthermore, because of the use of stored (lysed) blood samples, a calibrated pipette was used instead of the RDT kit’s original transfer device (“loop”). This could bypass possible errors in transfer volume, which may occur when performing the test in a field setting. Finally, the application of stringent interpretation criteria unfavourably influenced test outcomes: P. falciparum samples with pure gametocytaemia were included among the positive samples. Although meaningful in the scope of travel medicine
, it added to the false-negative results and decreased the sensitivity of SDFK90.
SDFK90 was previously evaluated in the third evaluation round of the World Health Organization (WHO) and the Foundation for Innovative New Diagnostics (FIND)
, in which detection of P. falciparum and P. vivax was assessed using diluted samples at fixed parasite densities. The detection rate for P. falciparum was 87.9% and 100% at low (200/μl) and high (2,000/μl or 5,000/μl) parasite densities respectively. In the present study, even at parasite densities <200/μl sensitivity already reached 97.2%.
PfHRP2 detection has advantages over the use of Pf-pLDH antigen detection. That is, PfHRP2-detecting RDTs are generally reported to be more heat resistant, although some Pf-pLDH-detecting RDTs do have extended temperature stabilities
[11, 14]. The heat stability testing of the WHO/FIND study confirmed resistance of the SDFK90 to temperatures up to 45°C
[11, 14–16]. Moreover, PfHRP2-detecting RDTs are generally reported to have lower sensitivity for P. falciparum diagnosis, especially at parasite densities ≤100/μl
[2, 11, 17–19].
On the other hand, detection of the Pf-pLDH antigen also has some advantages over PfHRP2 detection
. Unlike the PfHRP2 antigen, Pf-pLDH is not susceptible to the prozone effect, i. e., a missed or delayed diagnosis at high parasite densities, as was confirmed in the present study. Although rare in travel medicine, the consequences of prozone can lead to serious complications and the risks increase when laboratories rely on RDTs alone for diagnosis
[3, 20]. The detection of both Pf-pLDH and PfHRP2 will offer a back-up in case of a prozone effect. In addition, it will offer a back-up in sam-ples lacking the pfhrp2 gene
: 25.7%–41.0% of P. falciparum samples in the Peruvian Amazon lack the pfhrp2 gene which encodes PfHRP2
[5, 21]. All PfHRP2-detecting RDTs evaluated by Maltha et al. failed to correctly diagnose these samples, whereas SDFK90, like the Pf-pLDH-detecting RDTs, correctly identified the P. falciparum infections because of the presence of the Pf-pLDH test line
PfHRP2 is produced by asexual parasites and young gametocytes. It is expressed on the red blood cell membrane and readily diffuses in the plasma. Due to its slow clearance, PfHRP2 antigen persists in the bloodstream for up to several weeks after successful treatment of the infection
[22–24]. Pf-pLDH is an enzyme of the glycolytic pathway produced by asexual stages and gametocytes and its presence depends on living parasites. Pf-pLDH quickly disappears from the blood when parasites have been cleared
Although the Pf-pLDH antigen is rapidly cleared from the blood once treatment is initiated
[22–24], its use for treatment follow-up has been abandoned because of the persistence of gametocytes for up to two weeks after the start of the treatment
[26, 27]. On the other hand, artemisinin-based combination therapy results in low post-treatment gametocytaemia with Pf-pLDH antigen levels below the RDT detection threshold
. While the use of Pf-pLDH to follow-up treatment and detection of therapy resistance is still under debate and awaiting further study, it can be argued that the combination of PfHRP2 and Pf-pLDH in the SDFK90 could offer an opportunity for treatment follow-up.
As expected, the specificity of the Pf-pLDH line was higher compared to the PfHRP2 line
. This can be explained by the persistence of PfHRP2 antigen for up to several weeks after successful treatment. Due to the retrospective design of this study, clinical information explaining the reactions of the PfHRP2 line with three P. vivax samples was not available, but the identical positive result in PfHRP2 ELISA suggests PfHRP2 persistence after a previous P. falciparum infection.
Line intensities of Pf-pLDH were lower than those of PfHRP2, irrespective of parasite densities. In previous studies, the ITM team consistently found lower line intensities in pLDH test lines
[10, 11, 29–33]. In particular weak and faint line intensities are of concern, as they tend to be regarded as negative
. The observed shortcomings in instructions for the use and labelling of the kit’s package and contents were in line with observations made for other RDT kits
 and can easily be corrected.