Can rapid malaria tests detect a zoonotic malaria species even when they cannot identify it precisely?

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Plasmodium knowlesi causes zoonotic malaria across Southeast Asia. First-line diagnostic microscopy cannot reliably distinguish P. knowlesi from other human malaria species. Rapid diagnostic tests (RDTs) designed for P. falciparum and P. vivax are routinely used in areas where P. knowlesi also occurs, despite potential cross-reactivity involving species-specific antibody targets.

This diagnostic accuracy and laboratory detection-limit evaluation assessed ten RDTs: nine for detecting clinical P. knowlesi infections from Malaysia, and nine for assessing limits of detection (LoDs) using P. knowlesi (PkA1-H.1) and P. falciparum (Pf3D7) cultures. Targets included Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax-specific pLDH (Pv-pLDH).

Samples were collected before antimalarial treatment from 127 patients with microscopy-positive, PCR-confirmed P. knowlesi mono-infections. Median parasitaemia was 788/µL (IQR 247–5,565/µL). Compared with reference PCR, pan-pLDH sensitivity ranged from 50.6% (95% CI 39.6–61.5) for SD BIOLINE to 87.0% (95% CI 75.1–94.6) for First Response® and CareStart™ PAN. Pv-pLDH RDTs detected P. knowlesi with sensitivity up to 92.0% (95% CI 84.3–96.7%) for Biocredit™. At parasite counts ≥200/µL, the pan-pLDH test Standard Q and Pv-pLDH RDTs exceeded 95% sensitivity. RDT specificity against 26 PCR-confirmed negative controls was 100%.

For the six highest-performing RDTs, sensitivity did not differ significantly between samples taken before and after antimalarial treatment; the median post-treatment sampling time was 3 hours. Parasite ring stages were present in 30% of pretreatment samples. Ring-stage proportions, averaging 1.9%, were inversely correlated with test positivity for Biocredit™ and two CareStart™ RDTs.

For cultured P. knowlesi, CareStart™ PAN had the lowest LoD at 25 parasites/µL; other pan-pLDH tests had LoDs ranging from 98 to >2000 parasites/µL. The Pv-pLDH LoD for P. knowlesi was 49 parasites/µL. P. falciparum-pLDH and histidine-rich-protein-2 channels did not react with P. knowlesi.

The researchers concluded that selected RDTs showed sufficient performance to detect all human malaria species, including P. knowlesi, in co-endemic areas where microscopy is unavailable, particularly at higher parasite counts. However, these tests cannot reliably distinguish among non-falciparum malaria species.