First field evaluation of novel LDH- and HRP2-based rapid tests for Plasmodium vivax and Plasmodium falciparum malaria diagnosis

Background: Rapid diagnostic tests (RDTs) are crucial for malaria diagnosis. Where Plasmodium falciparum and Plasmodium vivax are co-endemic, and where P. falciparum hrp2/3 deletions are frequent, RDTs need to detect either species, and P. falciparum using additional antigens to HRP2, such as LDH. Methods: Clinical patients presenting for malaria diagnosis in southern Ethiopia were enrolled and tested by microscopy at the health center and by four different RDTs: (i) BIOCREDIT Malaria Ag Pf (cHL) with a line combining HRP2 and LDH for P. falciparum, (ii) BIOCREDIT Malaria Ag Pf/Pv (cHL/L) with one line combining HRP2 and LDH for P. falciparum and one with LDH for P. vivax, (iii) Bioline Malaria Ag Pf/Pf/Pv with separate lines for HRP2 and LDH for P. falciparum, and LDH for P. vivax, and (iv) First Response with an HRP2 line for P. falciparum and a LDH line for P. vivax. The two BIOCREDIT RDTs had not previously been tested in the field. qPCR and expert microscopy were conducted as reference tests. P. falciparum positive samples were typed for hrp2/3 deletion. Results: Among 708 patients included in the final analysis, 46.0% were positive by qPCR (77 P. falciparum mono-infections, 198 P. vivax mono-infections, and 51 mixed infections). Strong agreement was observed between results of the different RDTs, with no significant differences in sensitivity. At densities >20 parasites/µL by qPCR, all RDTs reached sensitivities of >96% for P. falciparum, compared to 63% by health center microscopy, and for P. vivax all RDTs reached sensitivities of >92%, compared to 72% by health center microscopy. Specificity was > 99% for all P. falciparum RDTs and >98% for all P. vivax RDTs. Only 2/53 P. falciparum infections typed carried hrp2 and hrp3 deletions, both were detected by all LDH-based RDTs. Conclusions: Use of RDTs improves diagnostic accuracy compared to microscopy. The novel BIOCREDIT and Bioline RDTs show high sensitivity and specificity for P. falciparum and P. vivax diagnosis. Author summary: Rapid diagnostic tests (RDTs) are key for malaria control. In many countries in the Horn of Africa, Latin America, and the Asian Pacific, P. falciparum and P. vivax are co-endemic. Further, while the HRP2 protein is the most sensitive target for P. falciparum RDTs, it can be deleted from the parasite’s genome, resulting in false-negative tests. Where this is the case, RDTs need to be able to detect alternative proteins, such as LDH. Some RDTs can detect P. vivax, and P. falciparum HRP2 and LDH though three separate test lines. New RDTs combine P. falciparum HRP2 and LDH into a single test line. These RDTs might reduce errors in test interpretation, as a single line indicates a P. falciparum infection, rather than three possible results (HRP2 only, LDH only, or HRP2 and LDH). In this study, RDTs with two vs. three lines performed virtually identical among clinical patients in Ethiopia. Compared to qPCR, at densities above 20 parasites per µL of blood, they detected >96% of P. falciparum and >92% of P. vivax infections. All RDTs detected substantially more infections than microscopy which is still widely used in health centers.